Isolated human aminoacylase, nucleic acid molecules encoding human aminoacylase, and uses thereof

ABSTRACT

The present invention provides amino acid sequences of peptides that are encoded by genes within the human genome, the enzyme peptides of the present invention. The present invention specifically provides isolated peptide and nucleic acid molecules, methods of identifying orthologs and paralogs of the enzyme peptides, and methods of identifying modulators of the enzyme peptides.

FIELD OF THE INVENTION

[0001] The present invention is in the field of enzyme proteins that arerelated to the aminoacylase subfamily, recombinant DNA molecules, andprotein production. The present invention specifically provides novelpeptides and proteins that effect protein phosphorylation and nucleicacid molecules encoding such peptide and protein molecules, all of whichare useful in the development of human therapeutics and diagnosticcompositions and methods.

BACKGROUND OF THE INVENTION

[0002] Many human enzymes serve as targets for the action ofpharmaceutically active compounds. Several classes of human enzymes thatserve as such targets include helicase, steroid esterase and sulfatase,convertase, synthase, dehydrogenase, monoxygenase, transferase, kinase,glutanase, decarboxylase, isomerase and reductase. It is thereforeimportant in developing new pharmaceutical compounds to identify targetenzyme proteins that can be put into high-throughput screening formats.The present invention advances the state of the art by providing novelhuman drug target enzymes related to the aminoacylase subfamily.

[0003] The present invention has a substantial similarity toaminoacylase-1. Aminoacylase-1 (ACY1, EC 3.5.1.14), a new type ofmetalloprotein, is a cytosolic enzyme with a wide range of tissueexpression and has been postulated to function in the catabolism andsalvage of acylated amino acids. ACY-1 is more highly expressed inkidney than in liver. ACY1 has been assigned to chromosome 3p21, aregion reduced to homozygosity in small-cell lung cancer and renal cellcarcinoma, and shows a reduced or absent expression in small-cell lungcancer cell lines and tumors. For a review related to aminoacylase-1,see Miller et al., Genomics 1990 Sep.; 8(1):149-54, Mitta et al., JBiochem (Tokyo) 1992 Dec.; 112(6):737-42.

[0004] Enzyme proteins, particularly members of the aminoacylasesubfamily, are a major target for drug action and development.Accordingly, it is valuable to the field of pharmaceutical developmentto identify and characterize previously unknown members of thissubfamily of enzyme proteins. The present invention advances the stateof the art by providing previously unidentified human enzyme proteins,and the polynucleotides encoding them, that have homology to members ofthe aminoacylase subfamily. These novel compositions are useful in thediagnosis, prevention and treatment of biological processes associatedwith human diseases.

SUMMARY OF THE INVENTION

[0005] The present invention is based in part on the identification ofamino acid sequences of human enzyme peptides and proteins that arerelated to the aminoacylase subfamily, as well as allelic variants andother mammalian orthologs thereof. These unique peptide sequences, andnucleic acid sequences that encode these peptides, can be used as modelsfor the development of human therapeutic targets, aid in theidentification of therapeutic proteins, and serve as targets for thedevelopment of human therapeutic agents that modulate enzyme activity incells and tissues that express the enzyme. Experimental data as providedin FIG. 1 indicates expression in humans in the placenta, T cells from Tcell leukemia, ovary, brain, lung and leukocyte.

DESCRIPTION OF THE FIGURE SHEETS

[0006]FIG. 1 provides the nucleotide sequence of a cDNA moleculesequence that encodes the enzyme protein of the present invention. (SEQID NO: 1) In addition, structure and functional information is provided,such as ATG start, stop and tissue distribution, where available, thatallows one to readily determine specific uses of inventions based onthis molecular sequence. Experimental data as provided in FIG. 1indicates expression in humans in the placenta, T cells from T cellleukemia, ovary, brain, lung and leukocyte.

[0007]FIG. 2 provides the predicted amino acid sequence of the enzyme ofthe present invention. (SEQ ID NO: 2) In addition structure andfunctional information such as protein family, function, andmodification sites is provided where available, allowing one to readilydetermine specific uses of inventions based on this molecular sequence.

[0008]FIG. 3 provides genomic sequences that span the gene encoding theenzyme protein of the present invention. (SEQ ID NO: 3) In additionstructure and functional information, such as intron/exon structure,promoter location, etc., is provided where available, allowing one toreadily determine specific uses of inventions based on this molecularsequence. As illustrated in FIG. 3, SNPs were identified at 10 differentnucleotide positions.

DETAILED DESCRIPTION OF THE INVENTION

[0009] General Description

[0010] The present invention is based on the sequencing of the humangenome. During the sequencing and assembly of the human genome, analysisof the sequence information revealed previously unidentified fragmentsof the human genome that encode peptides that share structural and/orsequence homology to protein/peptide/domains identified andcharacterized within the art as being a enzyme protein or part of aenzyme protein and are related to the aminoacylase subfamily. Utilizingthese sequences, additional genomic sequences were assembled andtranscript and/or cDNA sequences were isolated and characterized. Basedon this analysis, the present invention provides amino acid sequences ofhuman enzyme peptides and proteins that are related to the aminoacylasesubfamily, nucleic acid sequences in the form of transcript sequences,cDNA sequences and/or genomic sequences that encode these enzymepeptides and proteins, nucleic acid variation (allelic information),tissue distribution of expression, and information about the closest artknown protein/peptide/domain that has structural or sequence homology tothe enzyme of the present invention.

[0011] In addition to being previously unknown, the peptides that areprovided in the present invention are selected based on their ability tobe used for the development of commercially important products andservices. Specifically, the present peptides are selected based onhomology and/or structural relatedness to known enzyme proteins of theaminoacylase subfamily and the expression pattern observed. Experimentaldata as provided in FIG. 1 indicates expression in humans in theplacenta, T cells from T cell leukemia, ovary, brain, lung andleukocyte. The art has clearly established the commercial importance ofmembers of this family of proteins and proteins that have expressionpatterns similar to that of the present gene. Some of the more specificfeatures of the peptides of the present invention, and the uses thereof,are described herein, particularly in the Background of the Inventionand in the annotation provided in the Figures, and/or are known withinthe art for each of the known aminoacylase family or subfamily of enzymeproteins.

[0012] Specific Embodiments

[0013] Peptide Molecules

[0014] The present invention provides nucleic acid sequences that encodeprotein molecules that have been identified as being members of theenzyme family of proteins and are related to the aminoacylase subfamily(protein sequences are provided in FIG. 2, transcript/cDNA sequences areprovided in FIG. 1 and genomic sequences are provided in FIG. 3). Thepeptide sequences provided in FIG. 2, as well as the obvious variantsdescribed herein, particularly allelic variants as identified herein andusing the information in FIG. 3, will be referred herein as the enzymepeptides of the present invention, enzyme peptides, or peptides/proteinsof the present invention.

[0015] The present invention provides isolated peptide and proteinmolecules that consist of, consist essentially of, or comprise the aminoacid sequences of the enzyme peptides disclosed in the FIG. 2, (encodedby the nucleic acid molecule shown in FIG. 1, transcript/cDNA or FIG. 3,genomic sequence), as well as all obvious variants of these peptidesthat are within the art to make and use. Some of these variants aredescribed in detail below.

[0016] As used herein, a peptide is said to be “isolated” or “purified”when it is substantially free of cellular material or free of chemicalprecursors or other chemicals. The peptides of the present invention canbe purified to homogeneity or other degrees of purity. The level ofpurification will be based on the intended use. The critical feature isthat the preparation allows for the desired function of the peptide,even if in the presence of considerable amounts of other components (thefeatures of an isolated nucleic acid molecule is discussed below).

[0017] In some uses, “substantially free of cellular material” includespreparations of the peptide having less than about 30% (by dry weight)other proteins (i.e., contaminating protein), less than about 20% otherproteins, less than about 10% other proteins, or less than about 5%other proteins. When the peptide is recombinantly produced, it can alsobe substantially free of culture medium, i.e., culture medium representsless than about 20% of the volume of the protein preparation.

[0018] The language “substantially free of chemical precursors or otherchemicals” includes preparations of the peptide in which it is separatedfrom chemical precursors or other chemicals that are involved in itssynthesis. In one embodiment, the language “substantially free ofchemical precursors or other chemicals” includes preparations of theenzyme peptide having less than about 30% (by dry weight) chemicalprecursors or other chemicals, less than about 20% chemical precursorsor other chemicals, less than about 10% chemical precursors or otherchemicals, or less than about 5% chemical precursors or other chemicals.

[0019] The isolated enzyme peptide can be purified from cells thatnaturally express it, purified from cells that have been altered toexpress it (recombinant), or synthesized using known protein synthesismethods. Experimental data as provided in FIG. 1 indicates expression inhumans in the placenta, T cells from T cell leukemia, ovary, brain, lungand leukocyte. For example, a nucleic acid molecule encoding the enzymepeptide is cloned into an expression vector, the expression vectorintroduced into a host cell and the protein expressed in the host cell.The protein can then be isolated from the cells by an appropriatepurification scheme using standard protein purification techniques. Manyof these techniques are described in detail below.

[0020] Accordingly, the present invention provides proteins that consistof the amino acid sequences provided in FIG. 2 (SEQ ID NO: 2), forexample, proteins encoded by the transcript/cDNA nucleic acid sequencesshown in FIG. 1 (SEQ ID NO: 1) and the genomic sequences provided inFIG. 3 (SEQ ID NO: 3). The amino acid sequence of such a protein isprovided in FIG. 2. A protein consists of an amino acid sequence whenthe amino acid sequence is the final amino acid sequence of the protein.

[0021] The present invention further provides proteins that consistessentially of the amino acid sequences provided in FIG. 2 (SEQ ID NO:2), for example, proteins encoded by the transcript/cDNA nucleic acidsequences shown in FIG. 1 (SEQ ID NO: 1) and the genomic sequencesprovided in FIG. 3 (SEQ ID NO: 3). A protein consists essentially of anamino acid sequence when such an amino acid sequence is present withonly a few additional amino acid residues, for example from about 1 toabout 100 or so additional residues, typically from 1 to about 20additional residues in the final protein.

[0022] The present invention further provides proteins that comprise theamino acid sequences provided in FIG. 2 (SEQ ID NO: 2), for example,proteins encoded by the transcript/cDNA nucleic acid sequences shown inFIG. 1 (SEQ ID NO: 1) and the genomic sequences provided in FIG. 3 (SEQID NO: 3). A protein comprises an amino acid sequence when the aminoacid sequence is at least part of the final amino acid sequence of theprotein. In such a fashion, the protein can be only the peptide or haveadditional amino acid molecules, such as amino acid residues (contiguousencoded sequence) that are naturally associated with it or heterologousamino acid residues/peptide sequences. Such a protein can have a fewadditional amino acid residues or can comprise several hundred or moreadditional amino acids. The preferred classes of proteins that arecomprised of the enzyme peptides of the present invention are thenaturally occurring mature proteins. A brief description of how varioustypes of these proteins can be made/isolated is provided below.

[0023] The enzyme peptides of the present invention can be attached toheterologous sequences to form chimeric or fusion proteins. Suchchimeric and fusion proteins comprise a enzyme peptide operativelylinked to a heterologous protein having an amino acid sequence notsubstantially homologous to the enzyme peptide. “Operatively linked”indicates that the enzyme peptide and the heterologous protein are fusedin-frame. The heterologous protein can be fused to the N-terminus orC-terminus of the enzyme peptide.

[0024] In some uses, the fusion protein does not affect the activity ofthe enzyme peptide per se. For example, the fusion protein can include,but is not limited to, enzymatic fusion proteins, for examplebeta-galactosidase fusions, yeast two-hybrid GAL fusions, poly-Hisfusions, MYC-tagged, HI-tagged and Ig fusions. Such fusion proteins,particularly poly-His fusions, can facilitate the purification ofrecombinant enzyme peptide. In certain host cells (e.g., mammalian hostcells), expression and/or secretion of a protein can be increased byusing a heterologous signal sequence.

[0025] A chimeric or fusion protein can be produced by standardrecombinant DNA techniques. For example, DNA fragments coding for thedifferent protein sequences are ligated together in-frame in accordancewith conventional techniques. In another embodiment, the fusion gene canbe synthesized by conventional techniques including automated DNAsynthesizers. Alternatively, PCR amplification of gene fragments can becarried out using anchor primers which give rise to complementaryoverhangs between two consecutive gene fragments which can subsequentlybe annealed and re-amplified to generate a chimeric gene sequence (seeAusubel et al., Current Protocols in Molecular Biology, 1992). Moreover,many expression vectors are commercially available that already encode afusion moiety (e.g., a GST protein). A enzyme peptide-encoding nucleicacid can be cloned into such an expression vector such that the fusionmoiety is linked in-frame to the enzyme peptide.

[0026] As mentioned above, the present invention also provides andenables obvious variants of the amino acid sequence of the proteins ofthe present invention, such as naturally occurring mature forms of thepeptide, allelic/sequence variants of the peptides, non-naturallyoccurring recombinantly derived variants of the peptides, and orthologsand paralogs of the peptides. Such variants can readily be generatedusing art-known techniques in the fields of recombinant nucleic acidtechnology and protein biochemistry. It is understood, however, thatvariants exclude any amino acid sequences disclosed prior to theinvention.

[0027] Such variants can readily be identified/made using moleculartechniques and the sequence information disclosed herein. Further, suchvariants can readily be distinguished from other peptides based onsequence and/or structural homology to the enzyme peptides of thepresent invention. The degree of homology/identity present will be basedprimarily on whether the peptide is a functional variant ornon-functional variant, the amount of divergence present in the paralogfamily and the evolutionary distance between the orthologs.

[0028] To determine the percent identity of two amino acid sequences ortwo nucleic acid sequences, the sequences are aligned for optimalcomparison purposes (e.g., gaps can be introduced in one or both of afirst and a second amino acid or nucleic acid sequence for optimalalignment and non-homologous sequences can be disregarded for comparisonpurposes). In a preferred embodiment, at least 30%, 40%, 50%, 60%, 70%,80%, or 90% or more of the length of a reference sequence is aligned forcomparison purposes. The amino acid residues or nucleotides atcorresponding amino acid positions or nucleotide positions are thencompared. When a position in the first sequence is occupied by the sameamino acid residue or nucleotide as the corresponding position in thesecond sequence, then the molecules are identical at that position (asused herein amino acid or nucleic acid “identity” is equivalent to aminoacid or nucleic acid “homology”). The percent identity between the twosequences is a function of the number of identical positions shared bythe sequences, taking into account the number of gaps, and the length ofeach gap, which need to be introduced for optimal alignment of the twosequences.

[0029] The comparison of sequences and determination of percent identityand similarity between two sequences can be accomplished using amathematical algorithm. (Computational Molecular Biology, Lesk, A. M.,ed., Oxford University Press, New York, 1988; Biocomputing: Informaticsand Genome Projects, Smith, D. W., ed., Academic Press, New York, 1993;Computer Analysis of Sequence Data, Part 1, Griffin, A. M., and Griffin,H. G., eds., Humana Press, New Jersey, 1994; Sequence Analysis inMolecular Biology, von Heinje, G., Academic Press, 1987; and SequenceAnalysis Primer, Gribskov, M. and Devereux, J., eds., M Stockton Press,New York, 1991). In a preferred embodiment, the percent identity betweentwo amino acid sequences is determined using the Needleman and Wunsch(J. Mol. Biol. (48):444-453 (1970)) algorithm which has beenincorporated into the GAP program in the GCG software package (availableat http://www.gcg.com), using either a Blossom 62 matrix or a PAM250matrix, and a gap weight of 16, 14, 12, 10, 8, 6, or 4 and a lengthweight of 1, 2, 3, 4, 5, or 6. In yet another preferred embodiment, thepercent identity between two nucleotide sequences is determined usingthe GAP program in the GCG software package (Devereux, J., et al.,Nucleic Acids Res. 12(1):387 (1984)) (available at http://www.gcg.com),using a NWSgapdna.CMP matrix and a gap weight of 40, 50, 60, 70, or 80and a length weight of 1, 2, 3, 4, 5, or 6. In another embodiment, thepercent identity between two amino acid or nucleotide sequences isdetermined using the algorithm of E. Myers and W. Miller (CABIOS,4:11-17 (1989)) which has been incorporated into the ALIGN program(version 2.0), using a PAM120 weight residue table, a gap length penaltyof 12 and a gap penalty of 4.

[0030] The nucleic acid and protein sequences of the present inventioncan further be used as a “query sequence” to perform a search againstsequence databases to, for example, identify other family members orrelated sequences. Such searches can be performed using the NBLAST andXBLAST programs (version 2.0) of Altschul, et al. (J. Mol. Biol.215:403-10 (1990)). BLAST nucleotide searches can be performed with theNBLAST program, score=100, wordlength=12 to obtain nucleotide sequenceshomologous to the nucleic acid molecules of the invention. BLAST proteinsearches can be performed with the XBLAST program, score=50,wordlength=3 to obtain amino acid sequences homologous to the proteinsof the invention. To obtain gapped alignments for comparison purposes,Gapped BLAST can be utilized as described in Altschul et al. (NucleicAcids Res. 25(17):3389-3402 (1997)). When utilizing BLAST and gappedBLAST programs, the default parameters of the respective programs (e.g.,XBLAST and NBLAST) can be used.

[0031] Full-length pre-processed forms, as well as mature processedforms, of proteins that comprise one of the peptides of the presentinvention can readily be identified as having complete sequence identityto one of the enzyme peptides of the present invention as well as beingencoded by the same genetic locus as the enzyme peptide provided herein.As indicated by the data presented in FIG. 3, the map position wasdetermined to be on chromosome 3 by ePCR.

[0032] Allelic variants of a enzyme peptide can readily be identified asbeing a human protein having a high degree (significant) of sequencehomology/identity to at least a portion of the enzyme peptide as well asbeing encoded by the same genetic locus as the enzyme peptide providedherein. Genetic locus can readily be determined based on the genomicinformation provided in FIG. 3, such as the genomic sequence mapped tothe reference human. As indicated by the data presented in FIG. 3, themap position was determined to be on chromosome 3 by ePCR. As usedherein, two proteins (or a region of the proteins) have significanthomology when the amino acid sequences are typically at least about70-80%, 80-90%, and more typically at least about 90-95% or morehomologous. A significantly homologous amino acid sequence, according tothe present invention, will be encoded by a nucleic acid sequence thatwill hybridize to a enzyme peptide encoding nucleic acid molecule understringent conditions as more fully described below.

[0033]FIG. 3 provides information on SNPs that have been found in thegene encoding the enzyme protein of the present invention. SNPs wereidentified at 10 different nucleotide positions in introns and regions5′ and 3′ of the ORF. Such SNPs in introns and outside the ORF mayaffect control/regulatory elements.

[0034] Paralogs of a enzyme peptide can readily be identified as havingsome degree of significant sequence homology/identity to at least aportion of the enzyme peptide, as being encoded by a gene from humans,and as having similar activity or function. Two proteins will typicallybe considered paralogs when the amino acid sequences are typically atleast about 60% or greater, and more typically at least about 70% orgreater homology through a given region or domain. Such paralogs will beencoded by a nucleic acid sequence that will hybridize to a enzymepeptide encoding nucleic acid molecule under moderate to stringentconditions as more fully described below.

[0035] Orthologs of a enzyme peptide can readily be identified as havingsome degree of significant sequence homology/identity to at least aportion of the enzyme peptide as well as being encoded by a gene fromanother organism. Preferred orthologs will be isolated from mammals,preferably primates, for the development of human therapeutic targetsand agents. Such orthologs will be encoded by a nucleic acid sequencethat will hybridize to a enzyme peptide encoding nucleic acid moleculeunder moderate to stringent conditions, as more fully described below,depending on the degree of relatedness of the two organisms yielding theproteins.

[0036] Non-naturally occurring variants of the enzyme peptides of thepresent invention can readily be generated using recombinant techniques.Such variants include, but are not limited to deletions, additions andsubstitutions in the amino acid sequence of the enzyme peptide. Forexample, one class of substitutions are conserved amino acidsubstitution. Such substitutions are those that substitute a given aminoacid in a enzyme peptide by another amino acid of like characteristics.Typically seen as conservative substitutions are the replacements, onefor another, among the aliphatic amino acids Ala, Val, Leu, and Ile;interchange of the hydroxyl residues Ser and Thr; exchange of the acidicresidues Asp and Glu; substitution between the amide residues Asn andGln; exchange of the basic residues Lys and Arg; and replacements amongthe aromatic residues Phe and Tyr. Guidance concerning which amino acidchanges are likely to be phenotypically silent are found in Bowie etal., Science 247:1306-1310 (1990).

[0037] Variant enzyme peptides can be fully functional or can lackfunction in one or more activities, e.g. ability to bind substrate,ability to phosphorylate substrate, ability to mediate signaling, etc.Fully functional variants typically contain only conservative variationor variation in non-critical residues or in non-critical regions. FIG. 2provides the result of protein analysis and can be used to identifycritical domains/regions. Functional variants can also containsubstitution of similar amino acids that result in no change or aninsignificant change in function. Alternatively, such substitutions maypositively or negatively affect function to some degree.

[0038] Non-functional variants typically contain one or morenon-conservative amino acid substitutions, deletions, insertions,inversions, or truncation or a substitution, insertion, inversion, ordeletion in a critical residue or critical region.

[0039] Amino acids that are essential for function can be identified bymethods known in the art, such as site-directed mutagenesis oralanine-scanning mutagenesis (Cunningham et al., Science 244:1081-1085(1989)), particularly using the results provided in FIG. 2. The latterprocedure introduces single alanine mutations at every residue in themolecule. The resulting mutant molecules are then tested for biologicalactivity such as enzyme activity or in assays such as an in vitroproliferative activity. Sites that are critical for bindingpartner/substrate binding can also be determined by structural analysissuch as crystallization, nuclear magnetic resonance or photoaffinitylabeling (Smith et al., J. Mol. Biol. 224:899-904 (1992); de Vos et al.Science 255:306-312 (1992)).

[0040] The present invention further provides fragments of the enzymepeptides, in addition to proteins and peptides that comprise and consistof such fragments, particularly those comprising the residues identifiedin FIG. 2. The fragments to which the invention pertains, however, arenot to be construed as encompassing fragments that may be disclosedpublicly prior to the present invention.

[0041] As used herein, a fragment comprises at least 8, 10, 12, 14, 16,or more contiguous amino acid residues from a enzyme peptide. Suchfragments can be chosen based on the ability to retain one or more ofthe biological activities of the enzyme peptide or could be chosen forthe ability to perform a function, e.g. bind a substrate or act as animmunogen. Particularly important fragments are biologically activefragments, peptides that are, for example, about 8 or more amino acidsin length. Such fragments will typically comprise a domain or motif ofthe enzyme peptide, e.g., active site, a transmembrane domain or asubstrate-binding domain. Further, possible fragments include, but arenot limited to, domain or motif containing fragments, soluble peptidefragments, and fragments containing immunogenic structures. Predicteddomains and functional sites are readily identifiable by computerprograms well known and readily available to those of skill in the art(e.g., PROSITE analysis). The results of one such analysis are providedin FIG. 2.

[0042] Polypeptides often contain amino acids other than the 20 aminoacids commonly referred to as the 20 naturally occurring amino acids.Further, many amino acids, including the terminal amino acids, may bemodified by natural processes, such as processing and otherpost-translational modifications, or by chemical modification techniqueswell known in the art. Common modifications that occur naturally inenzyme peptides are described in basic texts, detailed monographs, andthe research literature, and they are well known to those of skill inthe art (some of these features are identified in FIG. 2).

[0043] Known modifications include, but are not limited to, acetylation,acylation, ADP-ribosylation, amidation, covalent attachment of flavin,covalent attachment of a heme moiety, covalent attachment of anucleotide or nucleotide derivative, covalent attachment of a lipid orlipid derivative, covalent attachment of phosphotidylinositol,cross-linking, cyclization, disulfide bond formation, demethylation,formation of covalent crosslinks, formation of cystine, formation ofpyroglutamate, formylation, gamma carboxylation, glycosylation, GPIanchor formation, hydroxylation, iodination, methylation,myristoylation, oxidation, proteolytic processing, phosphorylation,prenylation, racemization, selenoylation, sulfation, transfer-RNAmediated addition of amino acids to proteins such as arginylation, andubiquitination.

[0044] Such modifications are well known to those of skill in the artand have been described in great detail in the scientific literature.Several particularly common modifications, glycosylation, lipidattachment, sulfation, gamma-carboxylation of glutamic acid residues,hydroxylation and ADP-ribosylation, for instance, are described in mostbasic texts, such as Proteins-Structure and Molecular Properties, 2ndEd., T. E. Creighton, W. H. Freeman and Company, New York (1993). Manydetailed reviews are available on this subject, such as by Wold, F.,Posttranslational Covalent Modification of Proteins, B. C. Johnson, Ed.,Academic Press, New York 1-12 (1983); Seifter et al. (Meth. Enzymol.182: 626-646 (1990)) and Rattan et al. (Ann. N.Y. Acad. Sci. 663:48-62(1992)).

[0045] Accordingly, the enzyme peptides of the present invention alsoencompass derivatives or analogs in which a substituted amino acidresidue is not one encoded by the genetic code, in which a substituentgroup is included, in which the mature enzyme peptide is fused withanother compound, such as a compound to increase the half-life of theenzyme peptide (for example, polyethylene glycol), or in which theadditional amino acids are fused to the mature enzyme peptide, such as aleader or secretory sequence or a sequence for purification of themature enzyme peptide or a pro-protein sequence.

[0046] Protein/Peptide Uses

[0047] The proteins of the present invention can be used in substantialand specific assays related to the functional information provided inthe Figures; to raise antibodies or to elicit another immune response;as a reagent (including the labeled reagent) in assays designed toquantitatively determine levels of the protein (or its binding partneror ligand) in biological fluids; and as markers for tissues in which thecorresponding protein is preferentially expressed (either constitutivelyor at a particular stage of tissue differentiation or development or ina disease state). Where the protein binds or potentially binds toanother protein or ligand (such as, for example, in a enzyme-effectorprotein interaction or enzyme-ligand interaction), the protein can beused to identify the binding partner/ligand so as to develop a system toidentify inhibitors of the binding interaction. Any or all of these usesare capable of being developed into reagent grade or kit format forcommercialization as commercial products.

[0048] Methods for performing the uses listed above are well known tothose skilled in the art. References disclosing such methods include“Molecular Cloning: A Laboratory Manual”, 2d ed., Cold Spring HarborLaboratory Press, Sambrook, J., E. F. Fritsch and T. Maniatis eds.,1989, and “Methods in Enzymology: Guide to Molecular CloningTechniques”, Academic Press, Berger, S. L. and A. R. Kimmel eds., 1987.

[0049] The potential uses of the peptides of the present invention arebased primarily on the source of the protein as well as the class/actionof the protein. For example, enzymes isolated from humans and theirhuman/mammalian orthologs serve as targets for identifying agents foruse in mammalian therapeutic applications, e.g. a human drug,particularly in modulating a biological or pathological response in acell or tissue that expresses the enzyme. Experimental data as providedin FIG. 1 indicates that the enzymes of the present invention areexpressed in humans in the in the placenta, T cells from T cellleukemia, ovary, brain, lung detected by a virtual northern blot. Inaddition, PCR-based tissue screening panels indicate expression inleukocyte. A large percentage of pharmaceutical agents are beingdeveloped that modulate the activity of enzyme proteins, particularlymembers of the aminoacylase subfamily (see Background of the Invention).The structural and functional information provided in the Background andFigures provide specific and substantial uses for the molecules of thepresent invention, particularly in combination with the expressioninformation provided in FIG. 1. Experimental data as provided in FIG. 1indicates expression in humans in the placenta, T cells from T cellleukemia, ovary, brain, lung and leukocyte. Such uses can readily bedetermined using the information provided herein, that which is known inthe art, and routine experimentation.

[0050] The proteins of the present invention (including variants andfragments that may have been disclosed prior to the present invention)are useful for biological assays related to enzymes that are related tomembers of the aminoacylase subfamily. Such assays involve any of theknown enzyme functions or activities or properties useful for diagnosisand treatment of enzyme-related conditions that are specific for thesubfamily of enzymes that the one of the present invention belongs to,particularly in cells and tissues that express the enzyme. Experimentaldata as provided in FIG. 1 indicates that the enzymes of the presentinvention are expressed in humans in the in the placenta, T cells from Tcell leukemia, ovary, brain, lung detected by a virtual northern blot.In addition, PCR-based tissue screening panels indicate expression inleukocyte.

[0051] The proteins of the present invention are also useful in drugscreening assays, in cell-based or cell-free systems. Cell-based systemscan be native, i.e., cells that normally express the enzyme, as a biopsyor expanded in cell culture. Experimental data as provided in FIG. 1indicates expression in humans in the placenta, T cells from T cellleukemia, ovary, brain, lung and leukocyte. In an alternate embodiment,cell-based assays involve recombinant host cells expressing the enzymeprotein.

[0052] The polypeptides can be used to identify compounds that modulateenzyme activity of the protein in its natural state or an altered formthat causes a specific disease or pathology associated with the enzyme.Both the enzymes of the present invention and appropriate variants andfragments can be used in high-throughput screens to assay candidatecompounds for the ability to bind to the enzyme. These compounds can befurther screened against a functional enzyme to determine the effect ofthe compound on the enzyme activity. Further, these compounds can betested in animal or invertebrate systems to determineactivity/effectiveness. Compounds can be identified that activate(agonist) or inactivate (antagonist) the enzyme to a desired degree.

[0053] Further, the proteins of the present invention can be used toscreen a compound for the ability to stimulate or inhibit interactionbetween the enzyme protein and a molecule that normally interacts withthe enzyme protein, e.g. a substrate or a component of the signalpathway that the enzyme protein normally interacts (for example, anotherenzyme). Such assays typically include the steps of combining the enzymeprotein with a candidate compound under conditions that allow the enzymeprotein, or fragment, to interact with the target molecule, and todetect the formation of a complex between the protein and the target orto detect the biochemical consequence of the interaction with the enzymeprotein and the target, such as any of the associated effects of signaltransduction such as protein phosphorylation, cAMP turnover, andadenylate cyclase activation, etc.

[0054] Candidate compounds include, for example, 1) peptides such assoluble peptides, including Ig-tailed fusion peptides and members ofrandom peptide libraries (see, e.g., Lam et al., Nature 354:82-84(1991); Houghten et al., Nature 354:84-86 (1991)) and combinatorialchemistry-derived molecular libraries made of D- and/or L-configurationamino acids; 2) phosphopeptides (e.g., members of random and partiallydegenerate, directed phosphopeptide libraries, see, e.g., Songyang etal., Cell 72:767-778 (1993)); 3) antibodies (e.g., polyclonal,monoclonal, humanized, anti-idiotypic, chimeric, and single chainantibodies as well as Fab, F(ab′)₂, Fab expression library fragments,and epitope-binding fragments of antibodies); and 4) small organic andinorganic molecules (e.g., molecules obtained from combinatorial andnatural product libraries).

[0055] One candidate compound is a soluble fragment of the receptor thatcompetes for substrate binding. Other candidate compounds include mutantenzymes or appropriate fragments containing mutations that affect enzymefunction and thus compete for substrate. Accordingly, a fragment thatcompetes for substrate, for example with a higher affinity, or afragment that binds substrate but does not allow release, is encompassedby the invention.

[0056] The invention further includes other end point assays to identifycompounds that modulate (stimulate or inhibit) enzyme activity. Theassays typically involve an assay of events in the signal transductionpathway that indicate enzyme activity. Thus, the phosphorylation of asubstrate, activation of a protein, a change in the expression of genesthat are up- or down-regulated in response to the enzyme proteindependent signal cascade can be assayed.

[0057] Any of the biological or biochemical functions mediated by theenzyme can be used as an endpoint assay. These include all of thebiochemical or biochemical/biological events described herein, in thereferences cited herein, incorporated by reference for these endpointassay targets, and other functions known to those of ordinary skill inthe art or that can be readily identified using the information providedin the Figures, particularly FIG. 2. Specifically, a biological functionof a cell or tissues that expresses the enzyme can be assayed.Experimental data as provided in FIG. 1 indicates that the enzymes ofthe present invention are expressed in humans in the in the placenta, Tcells from T cell leukemia, ovary, brain, lung detected by a virtualnorthern blot. In addition, PCR-based tissue screening panels indicateexpression in leukocyte.

[0058] Binding and/or activating compounds can also be screened by usingchimeric enzyme proteins in which the amino terminal extracellulardomain, or parts thereof, the entire transmembrane domain or subregions,such as any of the seven transmembrane segments or any of theintracellular or extracellular loops and the carboxy terminalintracellular domain, or parts thereof, can be replaced by heterologousdomains or subregions. For example, a substrate-binding region can beused that interacts with a different substrate then that which isrecognized by the native enzyme. Accordingly, a different set of signaltransduction components is available as an end-point assay foractivation. This allows for assays to be performed in other than thespecific host cell from which the enzyme is derived.

[0059] The proteins of the present invention are also useful incompetition binding assays in methods designed to discover compoundsthat interact with the enzyme (e.g. binding partners and/or ligands).Thus, a compound is exposed to a enzyme polypeptide under conditionsthat allow the compound to bind or to otherwise interact with thepolypeptide. Soluble enzyme polypeptide is also added to the mixture. Ifthe test compound interacts with the soluble enzyme polypeptide, itdecreases the amount of complex formed or activity from the enzymetarget. This type of assay is particularly useful in cases in whichcompounds are sought that interact with specific regions of the enzyme.Thus, the soluble polypeptide that competes with the target enzymeregion is designed to contain peptide sequences corresponding to theregion of interest.

[0060] To perform cell free drug screening assays, it is sometimesdesirable to immobilize either the enzyme protein, or fragment, or itstarget molecule to facilitate separation of complexes from uncomplexedforms of one or both of the proteins, as well as to accommodateautomation of the assay.

[0061] Techniques for immobilizing proteins on matrices can be used inthe drug screening assays. In one embodiment, a fusion protein can beprovided which adds a domain that allows the protein to be bound to amatrix. For example, glutathione-S-transferase fusion proteins can beadsorbed onto glutathione sepharose beads (Sigma Chemical, St. Louis,Mo.) or glutathione derivatized microtiter plates, which are thencombined with the cell lysates (e.g., ³⁵S-labeled) and the candidatecompound, and the mixture incubated under conditions conducive tocomplex formation (e.g., at physiological conditions for salt and pH).Following incubation, the beads are washed to remove any unbound label,and the matrix immobilized and radiolabel determined directly, or in thesupernatant after the complexes are dissociated. Alternatively, thecomplexes can be dissociated from the matrix, separated by SDS-PAGE, andthe level of enzyme-binding protein found in the bead fractionquantitated from the gel using standard electrophoretic techniques. Forexample, either the polypeptide or its target molecule can beimmobilized utilizing conjugation of biotin and streptavidin usingtechniques well known in the art. Alternatively, antibodies reactivewith the protein but which do not interfere with binding of the proteinto its target molecule can be derivatized to the wells of the plate, andthe protein trapped in the wells by antibody conjugation. Preparationsof a enzyme-binding protein and a candidate compound are incubated inthe enzyme protein-presenting wells and the amount of complex trapped inthe well can be quantitated. Methods for detecting such complexes, inaddition to those described above for the GST-immobilized complexes,include immunodetection of complexes using antibodies reactive with theenzyme protein target molecule, or which are reactive with enzymeprotein and compete with the target molecule, as well as enzyme-linkedassays which rely on detecting an enzymatic activity associated with thetarget molecule.

[0062] Agents that modulate one of the enzymes of the present inventioncan be identified using one or more of the above assays, alone or incombination. It is generally preferable to use a cell-based or cell freesystem first and then confirm activity in an animal or other modelsystem. Such model systems are well known in the art and can readily beemployed in this context.

[0063] Modulators of enzyme protein activity identified according tothese drug screening assays can be used to treat a subject with adisorder mediated by the enzyme pathway, by treating cells or tissuesthat express the enzyme. Experimental data as provided in FIG. 1indicates expression in humans in the placenta, T cells from T cellleukemia, ovary, brain, lung and leukocyte. These methods of treatmentinclude the steps of administering a modulator of enzyme activity in apharmaceutical composition to a subject in need of such treatment, themodulator being identified as described herein.

[0064] In yet another aspect of the invention, the enzyme proteins canbe used as “bait proteins” in a two-hybrid assay or three-hybrid assay(see, e.g., U.S. Pat. No. 5,283,317; Zervos et al. (1993) Cell72:223-232; Madura et al. (1993) J. Biol. Chem. 268:12046-12054; Bartelet al. (1993) Biotechniques 14:920-924; Iwabuchi et al. (1993) Oncogene8:1693-1696; and Brent WO94/10300), to identify other proteins, whichbind to or interact with the enzyme and are involved in enzyme activity.Such enzyme-binding proteins are also likely to be involved in thepropagation of signals by the enzyme proteins or enzyme targets as, forexample, downstream elements of a enzyme-mediated signaling pathway.Alternatively, such enzyme-binding proteins are likely to be enzymeinhibitors.

[0065] The two-hybrid system is based on the modular nature of mosttranscription factors, which consist of separable DNA-binding andactivation domains. Briefly, the assay utilizes two different DNAconstructs. In one construct, the gene that codes for a enzyme proteinis fused to a gene encoding the DNA binding domain of a knowntranscription factor (e.g., GAL-4). In the other construct, a DNAsequence, from a library of DNA sequences, that encodes an unidentifiedprotein (“prey” or “sample”) is fused to a gene that codes for theactivation domain of the known transcription factor. If the “bait” andthe “prey” proteins are able to interact, in vivo, forming aenzyme-dependent complex, the DNA-binding and activation domains of thetranscription factor are brought into close proximity. This proximityallows transcription of a reporter gene (e.g., LacZ) which is operablylinked to a transcriptional regulatory site responsive to thetranscription factor. Expression of the reporter gene can be detectedand cell colonies containing the functional transcription factor can beisolated and used to obtain the cloned gene which encodes the proteinwhich interacts with the enzyme protein.

[0066] This invention further pertains to novel agents identified by theabove-described screening assays. Accordingly, it is within the scope ofthis invention to further use an agent identified as described herein inan appropriate animal model. For example, an agent identified asdescribed herein (e.g., a enzyme-modulating agent, an antisense enzymenucleic acid molecule, a enzyme-specific antibody, or a enzyme-bindingpartner) can be used in an animal or other model to determine theefficacy, toxicity, or side effects of treatment with such an agent.Alternatively, an agent identified as described herein can be used in ananimal or other model to determine the mechanism of action of such anagent. Furthermore, this invention pertains to uses of novel agentsidentified by the above-described screening assays for treatments asdescribed herein.

[0067] The enzyme proteins of the present invention are also useful toprovide a target for diagnosing a disease or predisposition to diseasemediated by the peptide. Accordingly, the invention provides methods fordetecting the presence, or levels of, the protein (or encoding mRNA) ina cell, tissue, or organism. Experimental data as provided in FIG. 1indicates expression in humans in the placenta, T cells from T cellleukemia, ovary, brain, lung and leukocyte. The method involvescontacting a biological sample with a compound capable of interactingwith the enzyme protein such that the interaction can be detected. Suchan assay can be provided in a single detection format or amulti-detection format such as an antibody chip array.

[0068] One agent for detecting a protein in a sample is an antibodycapable of selectively binding to protein. A biological sample includestissues, cells and biological fluids isolated from a subject, as well astissues, cells and fluids present within a subject.

[0069] The peptides of the present invention also provide targets fordiagnosing active protein activity, disease, or predisposition todisease, in a patient having a variant peptide, particularly activitiesand conditions that are known for other members of the family ofproteins to which the present one belongs. Thus, the peptide can beisolated from a biological sample and assayed for the presence of agenetic mutation that results in aberrant peptide. This includes aminoacid substitution, deletion, insertion, rearrangement, (as the result ofaberrant splicing events), and inappropriate post-translationalmodification. Analytic methods include altered electrophoretic mobility,altered tryptic peptide digest, altered enzyme activity in cell-based orcell-free assay, alteration in substrate or antibody-binding pattern,altered isoelectric point, direct amino acid sequencing, and any otherof the known assay techniques useful for detecting mutations in aprotein. Such an assay can be provided in a single detection format or amulti-detection format such as an antibody chip array.

[0070] In vitro techniques for detection of peptide include enzymelinked immunosorbent assays (ELISAs), Western blots,immunoprecipitations and immunofluorescence using a detection reagent,such as an antibody or protein binding agent. Alternatively, the peptidecan be detected in vivo in a subject by introducing into the subject alabeled anti-peptide antibody or other types of detection agent. Forexample, the antibody can be labeled with a radioactive marker whosepresence and location in a subject can be detected by standard imagingtechniques. Particularly useful are methods that detect the allelicvariant of a peptide expressed in a subject and methods which detectfragments of a peptide in a sample.

[0071] The peptides are also useful in pharmacogenomic analysis.Pharmacogenomics deal with clinically significant hereditary variationsin the response to drugs due to altered drug disposition and abnormalaction in affected persons. See, e.g., Eichelbaum, M. (Clin. Exp.Pharmacol. Physiol. 23(10-11):983-985 (1996)), and Linder, M. W. (Clin.Chem. 43(2):254-266 (1997)). The clinical outcomes of these variationsresult in severe toxicity of therapeutic drugs in certain individuals ortherapeutic failure of drugs in certain individuals as a result ofindividual variation in metabolism. Thus, the genotype of the individualcan determine the way a therapeutic compound acts on the body or the waythe body metabolizes the compound. Further, the activity of drugmetabolizing enzymes effects both the intensity and duration of drugaction. Thus, the pharmacogenomics of the individual permit theselection of effective compounds and effective dosages of such compoundsfor prophylactic or therapeutic treatment based on the individual'sgenotype. The discovery of genetic polymorphisms in some drugmetabolizing enzymes has explained why some patients do not obtain theexpected drug effects, show an exaggerated drug effect, or experienceserious toxicity from standard drug dosages. Polymorphisms can beexpressed in the phenotype of the extensive metabolizer and thephenotype of the poor metabolizer. Accordingly, genetic polymorphism maylead to allelic protein variants of the enzyme protein in which one ormore of the enzyme functions in one population is different from thosein another population. The peptides thus allow a target to ascertain agenetic predisposition that can affect treatment modality. Thus, in aligand-based treatment, polymorphism may give rise to amino terminalextracellular domains and/or other substrate-binding regions that aremore or less active in substrate binding, and enzyme activation.Accordingly, substrate dosage would necessarily be modified to maximizethe therapeutic effect within a given population containing apolymorphism. As an alternative to genotyping, specific polymorphicpeptides could be identified.

[0072] The peptides are also useful for treating a disordercharacterized by an absence of, inappropriate, or unwanted expression ofthe protein. Experimental data as provided in FIG. 1 indicatesexpression in humans in the placenta, T cells from T cell leukemia,ovary, brain, lung and leukocyte. Accordingly, methods for treatmentinclude the use of the enzyme protein or fragments.

[0073] Antibodies

[0074] The invention also provides antibodies that selectively bind toone of the peptides of the present invention, a protein comprising sucha peptide, as well as variants and fragments thereof. As used herein, anantibody selectively binds a target peptide when it binds the targetpeptide and does not significantly bind to unrelated proteins. Anantibody is still considered to selectively bind a peptide even if italso binds to other proteins that are not substantially homologous withthe target peptide so long as such proteins share homology with afragment or domain of the peptide target of the antibody. In this case,it would be understood that antibody binding to the peptide is stillselective despite some degree of cross-reactivity.

[0075] As used herein, an antibody is defined in terms consistent withthat recognized within the art: they are multi-subunit proteins producedby a mammalian organism in response to an antigen challenge. Theantibodies of the present invention include polyclonal antibodies andmonoclonal antibodies, as well as fragments of such antibodies,including, but not limited to, Fab or F(ab′)₂, and Fv fragments.

[0076] Many methods are known for generating and/or identifyingantibodies to a given target peptide. Several such methods are describedby Harlow, Antibodies, Cold Spring Harbor Press, (1989).

[0077] In general, to generate antibodies, an isolated peptide is usedas an immunogen and is administered to a mammalian organism, such as arat, rabbit or mouse. The full-length protein, an antigenic peptidefragment or a fusion protein can be used. Particularly importantfragments are those covering functional domains, such as the domainsidentified in FIG. 2, and domain of sequence homology or divergenceamongst the family, such as those that can readily be identified usingprotein alignment methods and as presented in the Figures.

[0078] Antibodies are preferably prepared from regions or discretefragments of the enzyme proteins. Antibodies can be prepared from anyregion of the peptide as described herein. However, preferred regionswill include those involved in function/activity and/or enzyme/bindingpartner interaction. FIG. 2 can be used to identify particularlyimportant regions while sequence alignment can be used to identifyconserved and unique sequence fragments.

[0079] An antigenic fragment will typically comprise at least 8contiguous amino acid residues. The antigenic peptide can comprise,however, at least 10, 12, 14, 16 or more amino acid residues. Suchfragments can be selected on a physical property, such as fragmentscorrespond to regions that are located on the surface of the protein,e.g., hydrophilic regions or can be selected based on sequenceuniqueness (see FIG. 2).

[0080] Detection on an antibody of the present invention can befacilitated by coupling (i.e., physically linking) the antibody to adetectable substance. Examples of detectable substances include variousenzymes, prosthetic groups, fluorescent materials, luminescentmaterials, bioluminescent materials, and radioactive materials. Examplesof suitable enzymes include horseradish peroxidase, alkalinephosphatase, β-galactosidase, or acetylcholinesterase; examples ofsuitable prosthetic group complexes include streptavidin/biotin andavidin/biotin; examples of suitable fluorescent materials includeumbelliferone, fluorescein, fluorescein isothiocyanate, rhodamine,dichlorotriazinylamine fluorescein, dansyl chloride or phycoerythrin; anexample of a luminescent material includes luminol; examples ofbioluminescent materials include luciferase, luciferin, and aequorin,and examples of suitable radioactive material include ¹²⁵I, ¹³¹I, ³⁵S or³H.

[0081] Antibody Uses

[0082] The antibodies can be used to isolate one of the proteins of thepresent invention by standard techniques, such as affinitychromatography or immunoprecipitation. The antibodies can facilitate thepurification of the natural protein from cells and recombinantlyproduced protein expressed in host cells. In addition, such antibodiesare useful to detect the presence of one of the proteins of the presentinvention in cells or tissues to determine the pattern of expression ofthe protein among various tissues in an organism and over the course ofnormal development. Experimental data as provided in FIG. 1 indicatesthat the enzymes of the present invention are expressed in humans in thein the placenta, T cells from T cell leukemia, ovary, brain, lungdetected by a virtual northern blot. In addition, PCR-based tissuescreening panels indicate expression in leukocyte. Further, suchantibodies can be used to detect protein in situ, in vitro, or in a celllysate or supernatant in order to evaluate the abundance and pattern ofexpression. Also, such antibodies can be used to assess abnormal tissuedistribution or abnormal expression during development or progression ofa biological condition. Antibody detection of circulating fragments ofthe full length protein can be used to identify turnover.

[0083] Further, the antibodies can be used to assess expression indisease states such as in active stages of the disease or in anindividual with a predisposition toward disease related to the protein'sfunction. When a disorder is caused by an inappropriate tissuedistribution, developmental expression, level of expression of theprotein, or expressed/processed form, the antibody can be preparedagainst the normal protein. Experimental data as provided in FIG. 1indicates expression in humans in the placenta, T cells from T cellleukemia, ovary, brain, lung and leukocyte. If a disorder ischaracterized by a specific mutation in the protein, antibodies specificfor this mutant protein can be used to assay for the presence of thespecific mutant protein.

[0084] The antibodies can also be used to assess normal and aberrantsubcellular localization of cells in the various tissues in an organism.Experimental data as provided in FIG. 1 indicates expression in humansin the placenta, T cells from T cell leukemia, ovary, brain, lung andleukocyte. The diagnostic uses can be applied, not only in genetictesting, but also in monitoring a treatment modality. Accordingly, wheretreatment is ultimately aimed at correcting expression level or thepresence of aberrant sequence and aberrant tissue distribution ordevelopmental expression, antibodies directed against the protein orrelevant fragments can be used to monitor therapeutic efficacy.

[0085] Additionally, antibodies are useful in pharmacogenomic analysis.Thus, antibodies prepared against polymorphic proteins can be used toidentify individuals that require modified treatment modalities. Theantibodies are also useful as diagnostic tools as an immunologicalmarker for aberrant protein analyzed by electrophoretic mobility,isoelectric point, tryptic peptide digest, and other physical assaysknown to those in the art.

[0086] The antibodies are also useful for tissue typing. Experimentaldata as provided in FIG. 1 indicates expression in humans in theplacenta, T cells from T cell leukemia, ovary, brain, lung andleukocyte. Thus, where a specific protein has been correlated withexpression in a specific tissue, antibodies that are specific for thisprotein can be used to identify a tissue type.

[0087] The antibodies are also useful for inhibiting protein function,for example, blocking the binding of the enzyme peptide to a bindingpartner such as a substrate. These uses can also be applied in atherapeutic context in which treatment involves inhibiting the protein'sfunction. An antibody can be used, for example, to block binding, thusmodulating (agonizing or antagonizing) the peptides activity. Antibodiescan be prepared against specific fragments containing sites required forfunction or against intact protein that is associated with a cell orcell membrane. See FIG. 2 for structural information relating to theproteins of the present invention.

[0088] The invention also encompasses kits for using antibodies todetect the presence of a protein in a biological sample. The kit cancomprise antibodies such as a labeled or labelable antibody and acompound or agent for detecting protein in a biological sample; meansfor determining the amount of protein in the sample; means for comparingthe amount of protein in the sample with a standard; and instructionsfor use. Such a kit can be supplied to detect a single protein orepitope or can be configured to detect one of a multitude of epitopes,such as in an antibody detection array. Arrays are described in detailbelow for nuleic acid arrays and similar methods have been developed forantibody arrays.

[0089] Nucleic Acid Molecules

[0090] The present invention further provides isolated nucleic acidmolecules that encode a enzyme peptide or protein of the presentinvention (cDNA, transcript and genomic sequence). Such nucleic acidmolecules will consist of, consist essentially of, or comprise anucleotide sequence that encodes one of the enzyme peptides of thepresent invention, an allelic variant thereof, or an ortholog or paralogthereof.

[0091] As used herein, an “isolated” nucleic acid molecule is one thatis separated from other nucleic acid present in the natural source ofthe nucleic acid. Preferably, an “isolated” nucleic acid is free ofsequences which naturally flank the nucleic acid (i.e., sequenceslocated at the 5′ and 3′ ends of the nucleic acid) in the genomic DNA ofthe organism from which the nucleic acid is derived. However, there canbe some flanking nucleotide sequences, for example up to about 5 KB, 4KB, 3 KB, 2 KB, or 1 KB or less, particularly contiguous peptideencoding sequences and peptide encoding sequences within the same genebut separated by introns in the genomic sequence. The important point isthat the nucleic acid is isolated from remote and unimportant flankingsequences such that it can be subjected to the specific manipulationsdescribed herein such as recombinant expression, preparation of probesand primers, and other uses specific to the nucleic acid sequences.

[0092] Moreover, an “isolated” nucleic acid molecule, such as atranscript/cDNA molecule, can be substantially free of other cellularmaterial, or culture medium when produced by recombinant techniques, orchemical precursors or other chemicals when chemically synthesized.However, the nucleic acid molecule can be fused to other coding orregulatory sequences and still be considered isolated.

[0093] For example, recombinant DNA molecules contained in a vector areconsidered isolated. Further examples of isolated DNA molecules includerecombinant DNA molecules maintained in heterologous host cells orpurified (partially or substantially) DNA molecules in solution.Isolated RNA molecules include in vivo or in vitro RNA transcripts ofthe isolated DNA molecules of the present invention. Isolated nucleicacid molecules according to the present invention further include suchmolecules produced synthetically.

[0094] Accordingly, the present invention provides nucleic acidmolecules that consist of the nucleotide sequence shown in FIGS. 1 or 3(SEQ ID NO: 1, transcript sequence and SEQ ID NO: 3, genomic sequence),or any nucleic acid molecule that encodes the protein provided in FIG.2, SEQ ID NO: 2. A nucleic acid molecule consists of a nucleotidesequence when the nucleotide sequence is the complete nucleotidesequence of the nucleic acid molecule.

[0095] The present invention further provides nucleic acid moleculesthat consist essentially of the nucleotide sequence shown in FIGS. 1 or3 (SEQ ID NO: 1, transcript sequence and SEQ ID NO: 3, genomicsequence), or any nucleic acid molecule that encodes the proteinprovided in FIG. 2, SEQ ID NO: 2. A nucleic acid molecule consistsessentially of a nucleotide sequence when such a nucleotide sequence ispresent with only a few additional nucleic acid residues in the finalnucleic acid molecule.

[0096] The present invention further provides nucleic acid moleculesthat comprise the nucleotide sequences shown in FIGS. 1 or 3 (SEQ ID NO:1, transcript sequence and SEQ ID NO: 3, genomic sequence), or anynucleic acid molecule that encodes the protein provided in FIG. 2, SEQID NO: 2. A nucleic acid molecule comprises a nucleotide sequence whenthe nucleotide sequence is at least part of the final nucleotidesequence of the nucleic acid molecule. In such a fashion, the nucleicacid molecule can be only the nucleotide sequence or have additionalnucleic acid residues, such as nucleic acid residues that are naturallyassociated with it or heterologous nucleotide sequences. Such a nucleicacid molecule can have a few additional nucleotides or can comprisesseveral hundred or more additional nucleotides. A brief description ofhow various types of these nucleic acid molecules can be readilymade/isolated is provided below.

[0097] In FIGS. 1 and 3, both coding and non-coding sequences areprovided. Because of the source of the present invention, humans genomicsequence (FIG. 3) and cDNA/transcript sequences (FIG. 1), the nucleicacid molecules in the Figures will contain genomic intronic sequences,5′ and 3′ non-coding sequences, gene regulatory regions and non-codingintergenic sequences. In general such sequence features are either notedin FIGS. 1 and 3 or can readily be identified using computational toolsknown in the art. As discussed below, some of the non-coding regions,particularly gene regulatory elements such as promoters, are useful fora variety of purposes, e.g. control of heterologous gene expression,target for identifying gene activity modulating compounds, and areparticularly claimed as fragments of the genomic sequence providedherein.

[0098] The isolated nucleic acid molecules can encode the mature proteinplus additional amino or carboxyl-terminal amino acids, or amino acidsinterior to the mature peptide (when the mature form has more than onepeptide chain, for instance). Such sequences may play a role inprocessing of a protein from precursor to a mature form, facilitateprotein trafficking, prolong or shorten protein half-life or facilitatemanipulation of a protein for assay or production, among other things.As generally is the case in situ, the additional amino acids may beprocessed away from the mature protein by cellular enzymes.

[0099] As mentioned above, the isolated nucleic acid molecules include,but are not limited to, the sequence encoding the enzyme peptide alone,the sequence encoding the mature peptide and additional codingsequences, such as a leader or secretory sequence (e.g., a pre-pro orpro-protein sequence), the sequence encoding the mature peptide, with orwithout the additional coding sequences, plus additional non-codingsequences, for example introns and non-coding 5′ and 3′ sequences suchas transcribed but non-translated sequences that play a role intranscription, mRNA processing (including splicing and polyadenylationsignals), ribosome binding and stability of mRNA. In addition, thenucleic acid molecule may be fused to a marker sequence encoding, forexample, a peptide that facilitates purification.

[0100] Isolated nucleic acid molecules can be in the form of RNA, suchas mRNA, or in the form DNA, including cDNA and genomic DNA obtained bycloning or produced by chemical synthetic techniques or by a combinationthereof. The nucleic acid, especially DNA, can be double-stranded orsingle-stranded. Single-stranded nucleic acid can be the coding strand(sense strand) or the non-coding strand (anti-sense strand).

[0101] The invention further provides nucleic acid molecules that encodefragments of the peptides of the present invention as well as nucleicacid molecules that encode obvious variants of the enzyme proteins ofthe present invention that are described above. Such nucleic acidmolecules may be naturally occurring, such as allelic variants (samelocus), paralogs (different locus), and orthologs (different organism),or may be constructed by recombinant DNA methods or by chemicalsynthesis. Such non-naturally occurring variants may be made bymutagenesis techniques, including those applied to nucleic acidmolecules, cells, or organisms. Accordingly, as discussed above, thevariants can contain nucleotide substitutions, deletions, inversions andinsertions. Variation can occur in either or both the coding andnon-coding regions. The variations can produce both conservative andnon-conservative amino acid substitutions.

[0102] The present invention further provides non-coding fragments ofthe nucleic acid molecules provided in FIGS. 1 and 3. Preferrednon-coding fragments include, but are not limited to, promotersequences, enhancer sequences, gene modulating sequences and genetermination sequences. Such fragments are useful in controllingheterologous gene expression and in developing screens to identifygene-modulating agents. A promoter can readily be identified as being 5′to the ATG start site in the genomic sequence provided in FIG. 3.

[0103] A fragment comprises a contiguous nucleotide sequence greaterthan 12 or more nucleotides. Further, a fragment could at least 30, 40,50, 100, 250 or 500 nucleotides in length. The length of the fragmentwill be based on its intended use. For example, the fragment can encodeepitope bearing regions of the peptide, or can be useful as DNA probesand primers. Such fragments can be isolated using the known nucleotidesequence to synthesize an oligonucleotide probe. A labeled probe canthen be used to screen a cDNA library, genomic DNA library, or mRNA toisolate nucleic acid corresponding to the coding region. Further,primers can be used in PCR reactions to clone specific regions of gene.

[0104] A probe/primer typically comprises substantially a purifiedoligonucleotide or oligonucleotide pair. The oligonucleotide typicallycomprises a region of nucleotide sequence that hybridizes understringent conditions to at least about 12, 20, 25, 40, 50 or moreconsecutive nucleotides.

[0105] Orthologs, homologs, and allelic variants can be identified usingmethods well known in the art. As described in the Peptide Section,these variants comprise a nucleotide sequence encoding a peptide that istypically 60-70%, 70-80%, 80-90%, and more typically at least about90-95% or more homologous to the nucleotide sequence shown in the Figuresheets or a fragment of this sequence. Such nucleic acid molecules canreadily be identified as being able to hybridize under moderate tostringent conditions, to the nucleotide sequence shown in the Figuresheets or a fragment of the sequence. Allelic variants can readily bedetermined by genetic locus of the encoding gene. As indicated by thedata presented in FIG. 3, the map position was determined to be onchromosome 3 by ePCR.

[0106]FIG. 3 provides information on SNPs that have been found in thegene encoding the enzyme protein of the present invention. SNPs wereidentified at 10 different nucleotide positions in introns and regions5′ and 3′ of the ORF. Such SNPs in introns and outside the ORF mayaffect control/regulatory elements.

[0107] As used herein, the term “hybridizes under stringent conditions”is intended to describe conditions for hybridization and washing underwhich nucleotide sequences encoding a peptide at least 60-70% homologousto each other typically remain hybridized to each other. The conditionscan be such that sequences at least about 60%, at least about 70%, or atleast about 80% or more homologous to each other typically remainhybridized to each other. Such stringent conditions are known to thoseskilled in the art and can be found in Current Protocols in MolecularBiology, John Wiley & Sons, N.Y. (1989), 6.3.1-6.3.6. One example ofstringent hybridization conditions are hybridization in 6×sodiumchloride/sodium citrate (SSC) at about 45C., followed by one or morewashes in 0.2×SSC, 0.1% SDS at 50-65C. Examples of moderate to lowstringency hybridization conditions are well known in the art.

[0108] Nucleic Acid Molecule Uses

[0109] The nucleic acid molecules of the present invention are usefulfor probes, primers, chemical intermediates, and in biological assays.The nucleic acid molecules are useful as a hybridization probe formessenger RNA, transcript/cDNA and genomic DNA to isolate full-lengthcDNA and genomic clones encoding the peptide described in FIG. 2 and toisolate cDNA and genomic clones that correspond to variants (alleles,orthologs, etc.) producing the same or related peptides shown in FIG. 2.As illustrated in FIG. 3, SNPs were identified at 10 differentnucleotide positions.

[0110] The probe can correspond to any sequence along the entire lengthof the nucleic acid molecules provided in the Figures. Accordingly, itcould be derived from 5′ noncoding regions, the coding region, and 3′noncoding regions. However, as discussed, fragments are not to beconstrued as encompassing fragments disclosed prior to the presentinvention.

[0111] The nucleic acid molecules are also useful as primers for PCR toamplify any given region of a nucleic acid molecule and are useful tosynthesize antisense molecules of desired length and sequence.

[0112] The nucleic acid molecules are also useful for constructingrecombinant vectors. Such vectors include expression vectors thatexpress a portion of, or all of, the peptide sequences. Vectors alsoinclude insertion vectors, used to integrate into another nucleic acidmolecule sequence, such as into the cellular genome, to alter in situexpression of a gene and/or gene product. For example, an endogenouscoding sequence can be replaced via homologous recombination with all orpart of the coding region containing one or more specifically introducedmutations.

[0113] The nucleic acid molecules are also useful for expressingantigenic portions of the proteins.

[0114] The nucleic acid molecules are also useful as probes fordetermining the chromosomal positions of the nucleic acid molecules bymeans of in situ hybridization methods. As indicated by the datapresented in FIG. 3, the map position was determined to be on chromosome3 by ePCR.

[0115] The nucleic acid molecules are also useful in making vectorscontaining the gene regulatory regions of the nucleic acid molecules ofthe present invention.

[0116] The nucleic acid molecules are also useful for designingribozymes corresponding to all, or a part, of the mRNA produced from thenucleic acid molecules described herein.

[0117] The nucleic acid molecules are also useful for making vectorsthat express part, or all, of the peptides.

[0118] The nucleic acid molecules are also useful for constructing hostcells expressing a part, or all, of the nucleic acid molecules andpeptides.

[0119] The nucleic acid molecules are also useful for constructingtransgenic animals expressing all, or a part, of the nucleic acidmolecules and peptides.

[0120] The nucleic acid molecules are also useful as hybridizationprobes for determining the presence, level, form and distribution ofnucleic acid expression. Experimental data as provided in FIG. 1indicates that the enzymes of the present invention are expressed inhumans in the in the placenta, T cells from T cell leukemia, ovary,brain, lung detected by a virtual northern blot. In addition, PCR-basedtissue screening panels indicate expression in leukocyte. Accordingly,the probes can be used to detect the presence of, or to determine levelsof, a specific nucleic acid molecule in cells, tissues, and inorganisms. The nucleic acid whose level is determined can be DNA or RNA.Accordingly, probes corresponding to the peptides described herein canbe used to assess expression and/or gene copy number in a given cell,tissue, or organism. These uses are relevant for diagnosis of disordersinvolving an increase or decrease in enzyme protein expression relativeto normal results.

[0121] In vitro techniques for detection of mRNA include Northernhybridizations and in situ hybridizations. In vitro techniques fordetecting DNA includes Southern hybridizations and in situhybridization.

[0122] Probes can be used as a part of a diagnostic test kit foridentifying cells or tissues that express a enzyme protein, such as bymeasuring a level of a enzyme-encoding nucleic acid in a sample of cellsfrom a subject e.g., mRNA or genomic DNA, or determining if a enzymegene has been mutated. Experimental data as provided in FIG. 1 indicatesthat the enzymes of the present invention are expressed in humans in thein the placenta, T cells from T cell leukemia, ovary, brain, lungdetected by a virtual northern blot. In addition, PCR-based tissuescreening panels indicate expression in leukocyte.

[0123] Nucleic acid expression assays are useful for drug screening toidentify compounds that modulate enzyme nucleic acid expression.

[0124] The invention thus provides a method for identifying a compoundthat can be used to treat a disorder associated with nucleic acidexpression of the enzyme gene, particularly biological and pathologicalprocesses that are mediated by the enzyme in cells and tissues thatexpress it. Experimental data as provided in FIG. 1 indicates expressionin humans in the placenta, T cells from T cell leukemia, ovary, brain,lung and leukocyte. The method typically includes assaying the abilityof the compound to modulate the expression of the enzyme nucleic acidand thus identifying a compound that can be used to treat a disordercharacterized by undesired enzyme nucleic acid expression. The assayscan be performed in cell-based and cell-free systems. Cell-based assaysinclude cells naturally expressing the enzyme nucleic acid orrecombinant cells genetically engineered to express specific nucleicacid sequences.

[0125] The assay for enzyme nucleic acid expression can involve directassay of nucleic acid levels, such as mRNA levels, or on collateralcompounds involved in the signal pathway. Further, the expression ofgenes that are up- or down-regulated in response to the enzyme proteinsignal pathway can also be assayed. In this embodiment the regulatoryregions of these genes can be operably linked to a reporter gene such asluciferase.

[0126] Thus, modulators of enzyme gene expression can be identified in amethod wherein a cell is contacted with a candidate compound and theexpression of mRNA determined. The level of expression of enzyme mRNA inthe presence of the candidate compound is compared to the level ofexpression of enzyme mRNA in the absence of the candidate compound. Thecandidate compound can then be identified as a modulator of nucleic acidexpression based on this comparison and be used, for example to treat adisorder characterized by aberrant nucleic acid expression. Whenexpression of mRNA is statistically significantly greater in thepresence of the candidate compound than in its absence, the candidatecompound is identified as a stimulator of nucleic acid expression. Whennucleic acid expression is statistically significantly less in thepresence of the candidate compound than in its absence, the candidatecompound is identified as an inhibitor of nucleic acid expression.

[0127] The invention further provides methods of treatment, with thenucleic acid as a target, using a compound identified through drugscreening as a gene modulator to modulate enzyme nucleic acid expressionin cells and tissues that express the enzyme. Experimental data asprovided in FIG. 1 indicates that the enzymes of the present inventionare expressed in humans in the in the placenta, T cells from T cellleukemia, ovary, brain, lung detected by a virtual northern blot. Inaddition, PCR-based tissue screening panels indicate expression inleukocyte. Modulation includes both up-regulation (i.e. activation oragonization) or down-regulation (suppression or antagonization) ornucleic acid expression.

[0128] Alternatively, a modulator for enzyme nucleic acid expression canbe a small molecule or drug identified using the screening assaysdescribed herein as long as the drug or small molecule inhibits theenzyme nucleic acid expression in the cells and tissues that express theprotein. Experimental data as provided in FIG. 1 indicates expression inhumans in the placenta, T cells from T cell leukemia, ovary, brain, lungand leukocyte.

[0129] The nucleic acid molecules are also useful for monitoring theeffectiveness of modulating compounds on the expression or activity ofthe enzyme gene in clinical trials or in a treatment regimen. Thus, thegene expression pattern can serve as a barometer for the continuingeffectiveness of treatment with the compound, particularly withcompounds to which a patient can develop resistance. The gene expressionpattern can also serve as a marker indicative of a physiologicalresponse of the affected cells to the compound. Accordingly, suchmonitoring would allow either increased administration of the compoundor the administration of alternative compounds to which the patient hasnot become resistant. Similarly, if the level of nucleic acid expressionfalls below a desirable level, administration of the compound could becommensurately decreased.

[0130] The nucleic acid molecules are also useful in diagnostic assaysfor qualitative changes in enzyme nucleic acid expression, andparticularly in qualitative changes that lead to pathology. The nucleicacid molecules can be used to detect mutations in enzyme genes and geneexpression products such as mRNA. The nucleic acid molecules can be usedas hybridization probes to detect naturally occurring genetic mutationsin the enzyme gene and thereby to determine whether a subject with themutation is at risk for a disorder caused by the mutation. Mutationsinclude deletion, addition, or substitution of one or more nucleotidesin the gene, chromosomal rearrangement, such as inversion ortransposition, modification of genomic DNA, such as aberrant methylationpatterns or changes in gene copy number, such as amplification.Detection of a mutated form of the enzyme gene associated with adysfunction provides a diagnostic tool for an active disease orsusceptibility to disease when the disease results from overexpression,underexpression, or altered expression of a enzyme protein.

[0131] Individuals carrying mutations in the enzyme gene can be detectedat the nucleic acid level by a variety of techniques. FIG. 3 providesinformation on SNPs that have been found in the gene encoding the enzymeprotein of the present invention. SNPs were identified at 10 differentnucleotide positions in introns and regions 5′ and 3′ of the ORF. SuchSNPs in introns and outside the ORF may affect control/regulatoryelements. As indicated by the data presented in FIG. 3, the map positionwas determined to be on chromosome 3 by ePCR. Genomic DNA can beanalyzed directly or can be amplified by using PCR prior to analysis.RNA or cDNA can be used in the same way. In some uses, detection of themutation involves the use of a probe/primer in a polymerase chainreaction (PCR) (see, e.g. U.S. Pat. Nos. 4,683,195 and 4,683,202), suchas anchor PCR or RACE PCR, or, alternatively, in a ligation chainreaction (LCR) (see, e.g., Landegran et al., Science 241:1077-1080(1988); and Nakazawa et al., PNAS 91:360-364 (1994)), the latter ofwhich can be particularly useful for detecting point mutations in thegene (see Abravaya et al., Nucleic Acids Res. 23:675-682 (1995)). Thismethod can include the steps of collecting a sample of cells from apatient, isolating nucleic acid (e.g., genomic, mRNA or both) from thecells of the sample, contacting the nucleic acid sample with one or moreprimers which specifically hybridize to a gene under conditions suchthat hybridization and amplification of the gene (if present) occurs,and detecting the presence or absence of an amplification product, ordetecting the size of the amplification product and comparing the lengthto a control sample. Deletions and insertions can be detected by achange in size of the amplified product compared to the normal genotype.Point mutations can be identified by hybridizing amplified DNA to normalRNA or antisense DNA sequences.

[0132] Alternatively, mutations in a enzyme gene can be directlyidentified, for example, by alterations in restriction enzyme digestionpatterns determined by gel electrophoresis.

[0133] Further, sequence-specific ribozymes (U.S. Pat. No. 5,498,531)can be used to score for the presence of specific mutations bydevelopment or loss of a ribozyme cleavage site. Perfectly matchedsequences can be distinguished from mismatched sequences by nucleasecleavage digestion assays or by differences in melting temperature.

[0134] Sequence changes at specific locations can also be assessed bynuclease protection assays such as RNase and S1 protection or thechemical cleavage method. Furthermore, sequence differences between amutant enzyme gene and a wild-type gene can be determined by direct DNAsequencing. A variety of automated sequencing procedures can be utilizedwhen performing the diagnostic assays (Naeve, C. W., (1995)Biotechniques 19:448), including sequencing by mass spectrometry (see,e.g., PCT International Publication No. WO 94/16101; Cohen et al., Adv.Chromatogr. 36:127-162 (1996); and Griffin et al., Appl. Biochem.Biotechnol. 38:147-159 (1993)).

[0135] Other methods for detecting mutations in the gene include methodsin which protection from cleavage agents is used to detect mismatchedbases in RNA/RNA or RNA/DNA duplexes (Myers et al., Science 230:1242(1985)); Cotton et al., PNAS 85:4397 (1988); Saleeba et al., Meth.Enzymol. 217:286-295 (1992)), electrophoretic mobility of mutant andwild type nucleic acid is compared (Orita et al., PNAS 86:2766 (1989);Cotton et al., Mutat. Res. 285:125-144 (1993); and Hayashi et al.,Genet. Anal. Tech. Appl. 9:73-79 (1992)), and movement of mutant orwild-type fragments in polyacrylamide gels containing a gradient ofdenaturant is assayed using denaturing gradient gel electrophoresis(Myers et al., Nature 313:495 (1985)). Examples of other techniques fordetecting point mutations include selective oligonucleotidehybridization, selective amplification, and selective primer extension.

[0136] The nucleic acid molecules are also useful for testing anindividual for a genotype that while not necessarily causing thedisease, nevertheless affects the treatment modality. Thus, the nucleicacid molecules can be used to study the relationship between anindividual's genotype and the individual's response to a compound usedfor treatment (pharmacogenomic relationship). Accordingly, the nucleicacid molecules described herein can be used to assess the mutationcontent of the enzyme gene in an individual in order to select anappropriate compound or dosage regimen for treatment. FIG. 3 providesinformation on SNPs that have been found in the gene encoding the enzymeprotein of the present invention. SNPs were identified at 10 differentnucleotide positions in introns and regions 5′ and 3′ of the ORF. SuchSNPs in introns and outside the ORF may affect control/regulatoryelements.

[0137] Thus nucleic acid molecules displaying genetic variations thataffect treatment provide a diagnostic target that can be used to tailortreatment in an individual. Accordingly, the production of recombinantcells and animals containing these polymorphisms allow effectiveclinical design of treatment compounds and dosage regimens.

[0138] The nucleic acid molecules are thus useful as antisenseconstructs to control enzyme gene expression in cells, tissues, andorganisms. A DNA antisense nucleic acid molecule is designed to becomplementary to a region of the gene involved in transcription,preventing transcription and hence production of enzyme protein. Anantisense RNA or DNA nucleic acid molecule would hybridize to the mRNAand thus block translation of mRNA into enzyme protein.

[0139] Alternatively, a class of antisense molecules can be used toinactivate mRNA in order to decrease expression of enzyme nucleic acid.Accordingly, these molecules can treat a disorder characterized byabnormal or undesired enzyme nucleic acid expression. This techniqueinvolves cleavage by means of ribozymes containing nucleotide sequencescomplementary to one or more regions in the mRNA that attenuate theability of the mRNA to be translated. Possible regions include codingregions and particularly coding regions corresponding to the catalyticand other functional activities of the enzyme protein, such as substratebinding.

[0140] The nucleic acid molecules also provide vectors for gene therapyin patients containing cells that are aberrant in enzyme geneexpression. Thus, recombinant cells, which include the patient's cellsthat have been engineered ex vivo and returned to the patient, areintroduced into an individual where the cells produce the desired enzymeprotein to treat the individual.

[0141] The invention also encompasses kits for detecting the presence ofa enzyme nucleic acid in a biological sample. Experimental data asprovided in FIG. 1 indicates that the enzymes of the present inventionare expressed in humans in the in the placenta, T cells from T cellleukemia, ovary, brain, lung detected by a virtual northern blot. Inaddition, PCR-based tissue screening panels indicate expression inleukocyte. For example, the kit can comprise reagents such as a labeledor labelable nucleic acid or agent capable of detecting enzyme nucleicacid in a biological sample; means for determining the amount of enzymenucleic acid in the sample; and means for comparing the amount of enzymenucleic acid in the sample with a standard. The compound or agent can bepackaged in a suitable container. The kit can further compriseinstructions for using the kit to detect enzyme protein mRNA or DNA.

[0142] Nucleic Acid Arrays

[0143] The present invention further provides nucleic acid detectionkits, such as arrays or microarrays of nucleic acid molecules that arebased on the sequence information provided in FIGS. 1 and 3 (SEQ ID NOS:1 and 3).

[0144] As used herein “Arrays” or “Microarrays” refers to an array ofdistinct polynucleotides or oligonucleotides synthesized on a substrate,such as paper, nylon or other type of membrane, filter, chip, glassslide, or any other suitable solid support. In one embodiment, themicroarray is prepared and used according to the methods described inU.S. Pat. No. 5,837,832, Chee et al, PCT application W095/11995 (Chee etal.), Lockhart, D. J. et al. (1996; Nat. Biotech. 14: 1675-1680) andSchena, M. et al. (1996; Proc. Natl. Acad. Sci. 93: 10614-10619), all ofwhich are incorporated herein in their entirety by reference. In otherembodiments, such arrays are produced by the methods described by Brownet al., U.S. Pat. No. 5,807,522.

[0145] The microarray or detection kit is preferably composed of a largenumber of unique, single-stranded nucleic acid sequences, usually eithersynthetic antisense oligonucleotides or fragments of cDNAs, fixed to asolid support. The oligonucleotides are preferably about 6-60nucleotides in length, more preferably 15-30 nucleotides in length, andmost preferably about 20-25 nucleotides in length. For a certain type ofmicroarray or detection kit, it may be preferable to useoligonucleotides that are only 7-20 nucleotides in length. Themicroarray or detection kit may contain oligonucleotides that cover theknown 5′, or 3′, sequence, sequential oligonucleotides which cover thefull length sequence; or unique oligonucleotides selected fromparticular areas along the length of the sequence. Polynucleotides usedin the microarray or detection kit may be oligonucleotides that arespecific to a gene or genes of interest.

[0146] In order to produce oligonucleotides to a known sequence for amicroarray or detection kit, the gene(s) of interest (or an ORFidentified from the contigs of the present invention) is typicallyexamined using a computer algorithm which starts at the 5′ or at the 3′end of the nucleotide sequence. Typical algorithms will then identifyoligomers of defined length that are unique to the gene, have a GCcontent within a range suitable for hybridization, and lack predictedsecondary structure that may interfere with hybridization. In certainsituations it may be appropriate to use pairs of oligonucleotides on amicroarray or detection kit. The “pairs” will be identical, except forone nucleotide that preferably is located in the center of the sequence.The second oligonucleotide in the pair (mismatched by one) serves as acontrol. The number of oligonucleotide pairs may range from two to onemillion. The oligomers are synthesized at designated areas on asubstrate using a light-directed chemical process. The substrate may bepaper, nylon or other type of membrane, filter, chip, glass slide or anyother suitable solid support.

[0147] In another aspect, an oligonucleotide may be synthesized on thesurface of the substrate by using a chemical coupling procedure and anink jet application apparatus, as described in PCT applicationW095/251116 (Baldeschweiler et al.) which is incorporated herein in itsentirety by reference. In another aspect, a “gridded” array analogous toa dot (or slot) blot may be used to arrange and link cDNA fragments oroligonucleotides to the surface of a substrate using a vacuum system,thermal, UV, mechanical or chemical bonding procedures. An array, suchas those described above, may be produced by hand or by using availabledevices (slot blot or dot blot apparatus), materials (any suitable solidsupport), and machines (including robotic instruments), and may contain8, 24, 96, 384, 1536, 6144 or more oligonucleotides, or any other numberbetween two and one million which lends itself to the efficient use ofcommercially available instrumentation.

[0148] In order to conduct sample analysis using a microarray ordetection kit, the RNA or DNA from a biological sample is made intohybridization probes. The mRNA is isolated, and cDNA is produced andused as a template to make antisense RNA (aRNA). The aRNA is amplifiedin the presence of fluorescent nucleotides, and labeled probes areincubated with the microarray or detection kit so that the probesequences hybridize to complementary oligonucleotides of the microarrayor detection kit. Incubation conditions are adjusted so thathybridization occurs with precise complementary matches or with variousdegrees of less complementarity. After removal of nonhybridized probes,a scanner is used to determine the levels and patterns of fluorescence.The scanned images are examined to determine degree of complementarityand the relative abundance of each oligonucleotide sequence on themicroarray or detection kit. The biological samples may be obtained fromany bodily fluids (such as blood, urine, saliva, phlegm, gastric juices,etc.), cultured cells, biopsies, or other tissue preparations. Adetection system may be used to measure the absence, presence, andamount of hybridization for all of the distinct sequencessimultaneously. This data may be used for large-scale correlationstudies on the sequences, expression patterns, mutations, variants, orpolymorphisms among samples.

[0149] Using such arrays, the present invention provides methods toidentify the expression of the enzyme proteins/peptides of the presentinvention. In detail, such methods comprise incubating a test samplewith one or more nucleic acid molecules and assaying for binding of thenucleic acid molecule with components within the test sample. Suchassays will typically involve arrays comprising many genes, at least oneof which is a gene of the present invention and or alleles of the enzymegene of the present invention. FIG. 3 provides information on SNPs thathave been found in the gene encoding the enzyme protein of the presentinvention. SNPs were identified at 10 different nucleotide positions inintrons and regions 5′ and 3′ of the ORF. Such SNPs in introns andoutside the ORF may affect control/regulatory elements.

[0150] Conditions for incubating a nucleic acid molecule with a testsample vary. Incubation conditions depend on the format employed in theassay, the detection methods employed, and the type and nature of thenucleic acid molecule used in the assay. One skilled in the art willrecognize that any one of the commonly available hybridization,amplification or array assay formats can readily be adapted to employthe novel fragments of the Human genome disclosed herein. Examples ofsuch assays can be found in Chard, T, An Introduction toRadioimmunoassay and Related Techniques, Elsevier Science Publishers,Amsterdam, The Netherlands (1986); Bullock, G. R. et al, Techniques inImmunocytochemistry, Academic Press, Orlando, Fla. Vol. 1 (1982), Vol. 2(1983), Vol. 3 (1985); Tijssen, P., Practice and Theory of EnzymeImmunoassays: Laboratory Techniques in Biochemistry and MolecularBiology, Elsevier Science Publishers, Amsterdam, The Netherlands (1985).

[0151] The test samples of the present invention include cells, proteinor membrane extracts of cells. The test sample used in theabove-described method will vary based on the assay format, nature ofthe detection method and the tissues, cells or extracts used as thesample to be assayed. Methods for preparing nucleic acid extracts or ofcells are well known in the art and can be readily be adapted in orderto obtain a sample that is compatible with the system utilized.

[0152] In another embodiment of the present invention, kits are providedwhich contain the necessary reagents to carry out the assays of thepresent invention.

[0153] Specifically, the invention provides a compartmentalized kit toreceive, in close confinement, one or more containers which comprises:(a) a first container comprising one of the nucleic acid molecules thatcan bind to a fragment of the Human genome disclosed herein; and (b) oneor more other containers comprising one or more of the following: washreagents, reagents capable of detecting presence of a bound nucleicacid.

[0154] In detail, a compartmentalized kit includes any kit in whichreagents are contained in separate containers. Such containers includesmall glass containers, plastic containers, strips of plastic, glass orpaper, or arraying material such as silica. Such containers allows oneto efficiently transfer reagents from one compartment to anothercompartment such that the samples and reagents are notcross-contaminated, and the agents or solutions of each container can beadded in a quantitative fashion from one compartment to another. Suchcontainers will include a container which will accept the test sample, acontainer which contains the nucleic acid probe, containers whichcontain wash reagents (such as phosphate buffered saline, Tris-buffers,etc.), and containers which contain the reagents used to detect thebound probe. One skilled in the art will readily recognize that thepreviously unidentified enzyme gene of the present invention can beroutinely identified using the sequence information disclosed herein canbe readily incorporated into one of the established kit formats whichare well known in the art, particularly expression arrays.

[0155] Vectors/host Cells

[0156] The invention also provides vectors containing the nucleic acidmolecules described herein. The term “vector” refers to a vehicle,preferably a nucleic acid molecule, which can transport the nucleic acidmolecules. When the vector is a nucleic acid molecule, the nucleic acidmolecules are covalently linked to the vector nucleic acid. With thisaspect of the invention, the vector includes a plasmid, single or doublestranded phage, a single or double stranded RNA or DNA viral vector, orartificial chromosome, such as a BAC, PAC, YAC, OR MAC.

[0157] A vector can be maintained in the host cell as anextrachromosomal element where it replicates and produces additionalcopies of the nucleic acid molecules. Alternatively, the vector mayintegrate into the host cell genome and produce additional copies of thenucleic acid molecules when the host cell replicates.

[0158] The invention provides vectors for the maintenance (cloningvectors) or vectors for expression (expression vectors) of the nucleicacid molecules. The vectors can function in prokaryotic or eukaryoticcells or in both (shuttle vectors).

[0159] Expression vectors contain cis-acting regulatory regions that areoperably linked in the vector to the nucleic acid molecules such thattranscription of the nucleic acid molecules is allowed in a host cell.The nucleic acid molecules can be introduced into the host cell with aseparate nucleic acid molecule capable of affecting transcription. Thus,the second nucleic acid molecule may provide a trans-acting factorinteracting with the cis-regulatory control region to allowtranscription of the nucleic acid molecules from the vector.Alternatively, a trans-acting factor may be supplied by the host cell.Finally, a trans-acting factor can be produced from the vector itself.It is understood, however, that in some embodiments, transcriptionand/or translation of the nucleic acid molecules can occur in acell-free system.

[0160] The regulatory sequence to which the nucleic acid moleculesdescribed herein can be operably linked include promoters for directingmRNA transcription. These include, but are not limited to, the leftpromoter from bacteriophage λ, the lac, TRP, and TAC promoters from E.coli, the early and late promoters from SV40, the CMV immediate earlypromoter, the adenovirus early and late promoters, and retroviruslong-terminal repeats.

[0161] In addition to control regions that promote transcription,expression vectors may also include regions that modulate transcription,such as repressor binding sites and enhancers. Examples include the SV40enhancer, the cytomegalovirus immediate early enhancer, polyomaenhancer, adenovirus enhancers, and retrovirus LTR enhancers.

[0162] In addition to containing sites for transcription initiation andcontrol, expression vectors can also contain sequences necessary fortranscription termination and, in the transcribed region a ribosomebinding site for translation. Other regulatory control elements forexpression include initiation and termination codons as well aspolyadenylation signals. The person of ordinary skill in the art wouldbe aware of the numerous regulatory sequences that are useful inexpression vectors. Such regulatory sequences are described, forexample, in Sambrook et al., Molecular Cloning: A Laboratory Manual.2nd. ed., Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y.,(1989).

[0163] A variety of expression vectors can be used to express a nucleicacid molecule. Such vectors include chromosomal, episomal, andvirus-derived vectors, for example vectors derived from bacterialplasmids, from bacteriophage, from yeast episomes, from yeastchromosomal elements, including yeast artificial chromosomes, fromviruses such as baculoviruses, papovaviruses such as SV40, Vacciniaviruses, adenoviruses, poxviruses, pseudorabies viruses, andretroviruses. Vectors may also be derived from combinations of thesesources such as those derived from plasmid and bacteriophage geneticelements, e.g. cosmids and phagemids. Appropriate cloning and expressionvectors for prokaryotic and eukaryotic hosts are described in Sambrooket al., Molecular Cloning: A Laboratory Manual. 2nd ed., Cold SpringHarbor Laboratory Press, Cold Spring Harbor, N.Y., (1989).

[0164] The regulatory sequence may provide constitutive expression inone or more host cells (i.e. tissue specific) or may provide forinducible expression in one or more cell types such as by temperature,nutrient additive, or exogenous factor such as a hormone or otherligand. A variety of vectors providing for constitutive and inducibleexpression in prokaryotic and eukaryotic hosts are well known to thoseof ordinary skill in the art.

[0165] The nucleic acid molecules can be inserted into the vectornucleic acid by well-known methodology. Generally, the DNA sequence thatwill ultimately be expressed is joined to an expression vector bycleaving the DNA sequence and the expression vector with one or morerestriction enzymes and then ligating the fragments together. Proceduresfor restriction enzyme digestion and ligation are well known to those ofordinary skill in the art.

[0166] The vector containing the appropriate nucleic acid molecule canbe introduced into an appropriate host cell for propagation orexpression using well-known techniques. Bacterial cells include, but arenot limited to, E. coli, Streptomyces, and Salmonella typhimurium.Eukaryotic cells include, but are not limited to, yeast, insect cellssuch as Drosophila, animal cells such as COS and CHO cells, and plantcells.

[0167] As described herein, it may be desirable to express the peptideas a fusion protein. Accordingly, the invention provides fusion vectorsthat allow for the production of the peptides. Fusion vectors canincrease the expression of a recombinant protein, increase thesolubility of the recombinant protein, and aid in the purification ofthe protein by acting for example as a ligand for affinity purification.A proteolytic cleavage site may be introduced at the junction of thefusion moiety so that the desired peptide can ultimately be separatedfrom the fusion moiety. Proteolytic enzymes include, but are not limitedto, factor Xa, thrombin, and enteroenzyme. Typical fusion expressionvectors include pGEX (Smith et al., Gene 67:31-40 (1988)), pMAL (NewEngland Biolabs, Beverly, Mass.) and pRIT5 (Pharmacia, Piscataway, N.J.)which fuse glutathione S-transferase (GST), maltose E binding protein,or protein A, respectively, to the target recombinant protein. Examplesof suitable inducible non-fusion E. coli expression vectors include pTrc(Amann et al., Gene 69:301-315 (1988)) and pET 11d (Studier et al., GeneExpression Technology: Methods in Enzymology 185:60-89 (1990)).

[0168] Recombinant protein expression can be maximized in host bacteriaby providing a genetic background wherein the host cell has an impairedcapacity to proteolytically cleave the recombinant protein. (Gottesman,S., Gene Expression Technology: Methods in Enzymology 185, AcademicPress, San Diego, Calif. (1990) 119-128). Alternatively, the sequence ofthe nucleic acid molecule of interest can be altered to providepreferential codon usage for a specific host cell, for example E. coli.(Wada et al., Nucleic Acids Res. 20:2111-2118 (1992)).

[0169] The nucleic acid molecules can also be expressed by expressionvectors that are operative in yeast. Examples of vectors for expressionin yeast e.g., S. cerevisiae include pYepSec1 (Baldari, et al., EMBO J.6:229-234 (1987)), pMFa (Kurjan et al., Cell 30:933-943(1982)), pJRY88(Schultz et al., Gene 54:113-123 (1987)), and pYES2 (InvitrogenCorporation, San Diego, Calif.).

[0170] The nucleic acid molecules can also be expressed in insect cellsusing, for example, baculovirus expression vectors. Baculovirus vectorsavailable for expression of proteins in cultured insect cells (e.g., Sf9cells) include the pAc series (Smith et al., Mol. Cell Biol. 3:2156-2165(1983)) and the pVL series (Lucklow et al., Virology 170:31-39 (1989)).

[0171] In certain embodiments of the invention, the nucleic acidmolecules described herein are expressed in mammalian cells usingmammalian expression vectors. Examples of mammalian expression vectorsinclude pCDM8 (Seed, B. Nature 329:840(1987)) and pMT2PC (Kaufmnan etal, EMBOJ 6:187-195 (1987)).

[0172] The expression vectors listed herein are provided by way ofexample only of the well-known vectors available to those of ordinaryskill in the art that would be useful to express the nucleic acidmolecules. The person of ordinary skill in the art would be aware ofother vectors suitable for maintenance propagation or expression of thenucleic acid molecules described herein. These are found for example inSambrook, J., Fritsh, E. F., and Maniatis, T. Molecular Cloning: ALaboratory Manual. 2nd, ed., Cold Spring Harbor Laboratory, Cold SpringHarbor Laboratory Press, Cold Spring Harbor, N.Y., 1989.

[0173] The invention also encompasses vectors in which the nucleic acidsequences described herein are cloned into the vector in reverseorientation, but operably linked to a regulatory sequence that permitstranscription of antisense RNA. Thus, an antisense transcript can beproduced to all, or to a portion, of the nucleic acid molecule sequencesdescribed herein, including both coding and non-coding regions.Expression of this antisense RNA is subject to each of the parametersdescribed above in relation to expression of the sense RNA (regulatorysequences, constitutive or inducible expression, tissue-specificexpression).

[0174] The invention also relates to recombinant host cells containingthe vectors described herein. Host cells therefore include prokaryoticcells, lower eukaryotic cells such as yeast, other eukaryotic cells suchas insect cells, and higher eukaryotic cells such as mammalian cells.

[0175] The recombinant host cells are prepared by introducing the vectorconstructs described herein into the cells by techniques readilyavailable to the person of ordinary skill in the art. These include, butare not limited to, calcium phosphate transfection,DEAE-dextran-mediated transfection, cationic lipid-mediatedtransfection, electroporation, transduction, infection, lipofection, andother techniques such as those found in Sambrook, et al. (MolecularCloning: A Laboratory Manual. 2nd, ed., Cold Spring Harbor Laboratory,Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y., 1989).

[0176] Host cells can contain more than one vector. Thus, differentnucleotide sequences can be introduced on different vectors of the samecell. Similarly, the nucleic acid molecules can be introduced eitheralone or with other nucleic acid molecules that are not related to thenucleic acid molecules such as those providing trans-acting factors forexpression vectors. When more than one vector is introduced into a cell,the vectors can be introduced independently, co-introduced or joined tothe nucleic acid molecule vector.

[0177] In the case of bacteriophage and viral vectors, these can beintroduced into cells as packaged or encapsulated virus by standardprocedures for infection and transduction. Viral vectors can bereplication-competent or replication-defective. In the case in whichviral replication is defective, replication will occur in host cellsproviding functions that complement the defects.

[0178] Vectors generally include selectable markers that enable theselection of the subpopulation of cells that contain the recombinantvector constructs. The marker can be contained in the same vector thatcontains the nucleic acid molecules described herein or may be on aseparate vector. Markers include tetracycline or ampicillin-resistancegenes for prokaryotic host cells and dihydrofolate reductase or neomycinresistance for eukaryotic host cells. However, any marker that providesselection for a phenotypic trait will be effective.

[0179] While the mature proteins can be produced in bacteria, yeast,mammalian cells, and other cells under the control of the appropriateregulatory sequences, cell-free transcription and translation systemscan also be used to produce these proteins using RNA derived from theDNA constructs described herein.

[0180] Where secretion of the peptide is desired, which is difficult toachieve with multi-transmembrane domain containing proteins such asenzymes, appropriate secretion signals are incorporated into the vector.The signal sequence can be endogenous to the peptides or heterologous tothese peptides.

[0181] Where the peptide is not secreted into the medium, which istypically the case with enzymes, the protein can be isolated from thehost cell by standard disruption procedures, including freeze thaw,sonication, mechanical disruption, use of lysing agents and the like.The peptide can then be recovered and purified by well-knownpurification methods including ammonium sulfate precipitation, acidextraction, anion or cationic exchange chromatography, phosphocellulosechromatography, hydrophobic-interaction chromatography, affinitychromatography, hydroxylapatite chromatography, lectin chromatography,or high performance liquid chromatography.

[0182] It is also understood that depending upon the host cell inrecombinant production of the peptides described herein, the peptidescan have various glycosylation patterns, depending upon the cell, ormaybe non-glycosylated as when produced in bacteria. In addition, thepeptides may include an initial modified methionine in some cases as aresult of a host-mediated process.

[0183] Uses of Vectors and Host Cells

[0184] The recombinant host cells expressing the peptides describedherein have a variety of uses. First, the cells are useful for producinga enzyme protein or peptide that can be further purified to producedesired amounts of enzyme protein or fragments. Thus, host cellscontaining expression vectors are useful for peptide production.

[0185] Host cells are also useful for conducting cell-based assaysinvolving the enzyme protein or enzyme protein fragments, such as thosedescribed above as well as other formats known in the art. Thus, arecombinant host cell expressing a native enzyme protein is useful forassaying compounds that stimulate or inhibit enzyme protein function.

[0186] Host cells are also useful for identifying enzyme protein mutantsin which these functions are affected. If the mutants naturally occurand give rise to a pathology, host cells containing the mutations areuseful to assay compounds that have a desired effect on the mutantenzyme protein (for example, stimulating or inhibiting function) whichmay not be indicated by their effect on the native enzyme protein.

[0187] Genetically engineered host cells can be further used to producenon-human transgenic animals. A transgenic animal is preferably amammal, for example a rodent, such as a rat or mouse, in which one ormore of the cells of the animal include a transgene. A transgene isexogenous DNA which is integrated into the genome of a cell from which atransgenic animal develops and which remains in the genome of the matureanimal in one or more cell types or tissues of the transgenic animal.These animals are useful for studying the function of a enzyme proteinand identifying and evaluating modulators of enzyme protein activity.Other examples of transgenic animals include non-human primates, sheep,dogs, cows, goats, chickens, and amphibians.

[0188] A transgenic animal can be produced by introducing nucleic acidinto the male pronuclei of a fertilized oocyte, e.g., by microinjection,retroviral infection, and allowing the oocyte to develop in apseudopregnant female foster animal. Any of the enzyme proteinnucleotide sequences can be introduced as a transgene into the genome ofa non-human animal, such as a mouse.

[0189] Any of the regulatory or other sequences useful in expressionvectors can form part of the transgenic sequence. This includes intronicsequences and polyadenylation signals, if not already included. Atissue-specific regulatory sequence(s) can be operably linked to thetransgene to direct expression of the enzyme protein to particularcells.

[0190] Methods for generating transgenic animals via embryo manipulationand microinjection, particularly animals such as mice, have becomeconventional in the art and are described, for example, in U.S. Pat.Nos. 4,736,866 and 4,870,009, both by Leder et al., U.S. Pat. No.4,873,191 by Wagner et al. and in Hogan, B., Manipulating the MouseEmbryo, (Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y.,1986). Similar methods are used for production of other transgenicanimals. A transgenic founder animal can be identified based upon thepresence of the transgene in its genome and/or expression of transgenicmRNA in tissues or cells of the animals. A transgenic founder animal canthen be used to breed additional animals carrying the transgene.Moreover, transgenic animals carrying a transgene can further be bred toother transgenic animals carrying other transgenes. A transgenic animalalso includes animals in which the entire animal or tissues in theanimal have been produced using the homologously recombinant host cellsdescribed herein.

[0191] In another embodiment, transgenic non-human animals can beproduced which contain selected systems that allow for regulatedexpression of the transgene. One example of such a system is thecre/loxP recombinase system of bacteriophage P1. For a description ofthe cre/loxP recombinase system, see, e.g., Lakso et al. PNAS89:6232-6236 (1992). Another example of a recombinase system is the FLPrecombinase system of S. cerevisiae (O'Gorman et al. Science251:1351-1355 (1991). If a cre/loxP recombinase system is used toregulate expression of the transgene, animals containing transgenesencoding both the Cre recombinase and a selected protein is required.Such animals can be provided through the construction of “double”transgenic animals, e.g., by mating two transgenic animals, onecontaining a transgene encoding a selected protein and the othercontaining a transgene encoding a recombinase.

[0192] Clones of the non-human transgenic animals described herein canalso be produced according to the methods described in Wilmut, I. et al.Nature 385:810-813 (1997) and PCT International Publication Nos. WO97/07668 and WO 97/07669. In brief, a cell, e.g., a somatic cell, fromthe transgenic animal can be isolated and induced to exit the growthcycle and enter G_(o) phase. The quiescent cell can then be fused, e.g.,through the use of electrical pulses, to an nucleated oocyte from ananimal of the same species from which the quiescent cell is isolated.The reconstructed oocyte is then cultured such that it develops tomorula or blastocyst and then transferred to pseudopregnant femalefoster animal. The offspring born of this female foster animal will be aclone of the animal from which the cell, e.g., the somatic cell, isisolated.

[0193] Transgenic animals containing recombinant cells that express thepeptides described herein are useful to conduct the assays describedherein in an in vivo context. Accordingly, the various physiologicalfactors that are present in vivo and that could effect substratebinding, enzyme protein activation, and signal transduction, may not beevident from in vitro cell-free or cell-based assays. Accordingly, it isuseful to provide non-human transgenic animals to assay in vivo enzymeprotein function, including substrate interaction, the effect ofspecific mutant enzyme proteins on enzyme protein function and substrateinteraction, and the effect of chimeric enzyme proteins. It is alsopossible to assess the effect of null mutations, that is, mutations thatsubstantially or completely eliminate one or more enzyme proteinfunctions.

[0194] All publications and patents mentioned in the above specificationare herein incorporated by reference. Various modifications andvariations of the described method and system of the invention will beapparent to those skilled in the art without departing from the scopeand spirit of the invention. Although the invention has been describedin connection with specific preferred embodiments, it should beunderstood that the invention as claimed should not be unduly limited tosuch specific embodiments. Indeed, various modifications of theabove-described modes for carrying out the invention which are obviousto those skilled in the field of molecular biology or related fields areintended to be within the scope of the following claims.

1 14 1 1445 DNA Homo sapiens 1 ctgcgacctc gcaggcgacc tcgctggaccctaagtccag gccacagtca gggaagggcg 60 ctgagaggcg agcgtgagcc cagcgacaggagagtgagct caccacgcgc agcgccatga 120 ccagcaaggg tcccgaggag gagcacccatcggtgacgct cttccgccag tacctgcgta 180 tccgcactgt ccagcccaag cctgactatggcaccaaccc tacactctcc tccatcttgc 240 tcaactccca cacggatgtg gtgcctgtcttcaaggaaca ttggagtcac gacccctttg 300 aggccttcaa ggattctgag ggctacatctatgccagggg tgcccaggac atgaagtgcg 360 tcagcatcca gtacctggaa gctgtgaggaggctgaaggt ggagggccac cggttcccca 420 gaaccatcca catgaccttt gtgcctgatgaggaggttgg gggtcaccaa ggcatggagc 480 tgttcgtgca gcggcctgag ttccacgccctgagggcagg ctttgccctg gatgagggca 540 tagccaatcc cactgatgcc ttcactgtcttttatagtga gcggagtccc tggtgggtgc 600 gggttaccag cactgggagg ccaggccatgcctcacgctt catggaggac acagcagcag 660 agaagctgca caaggttgta aactccatcctggcattccg ggagaaggaa tggcagaggc 720 tgcagtcaaa cccccacctg aaagaggggtccgtgacctc cgtgaacctg actaagctag 780 agggtggcgt ggcctataac gtgatacctgccaccatgag cgccagcttt gacttccgtg 840 tggcaccgga tgtggacttc aaggcttttgaggagcagct gcagagctgg tgccaggcag 900 ctggcgaggg ggtcacccta gagtttgctcagaagtggat gcacccccaa gtgacaccta 960 ctgatgactc aaacccttgg tgggcagcttttagccgggt ctgcaaggat atgaacctca 1020 ctctggagcc tgagatcatg cctgctgccactgacaaccg ctatatccgc gcggtggggg 1080 tcccagctct aggcttctca cccatgaaccgcacacctgt gctgctgcac gaccacgatg 1140 aacggctgca tgaggctgtg ttcctccgtggggtggacat atatacacgc ctgctgcctg 1200 cccttgccag tgtgcctgcc ctgcccagtgacagctgagc cctggaactc ctaaaccttt 1260 gcccctgggg cttccatccc aaccagtgccaaggacctcc tcttccccct tccaaataat 1320 aaagtctatg gacagggctg tctctgaagtactaacacaa aaaaaaaaaa aaaaaaaaaa 1380 aaaaaaaaaa aaaaaaaaaa aaaaaaaaaaaaaaaaaaaa aaaaaaaaaa aaaaaaaaaa 1440 aaaaa 1445 2 373 PRT Homo sapiens2 Met Thr Ser Lys Gly Pro Glu Glu Glu His Pro Ser Val Thr Leu Phe 1 5 1015 Arg Gln Tyr Leu Arg Ile Arg Thr Val Gln Pro Lys Pro Asp Tyr Gly 20 2530 Thr Asn Pro Thr Leu Ser Ser Ile Leu Leu Asn Ser His Thr Asp Val 35 4045 Val Pro Val Phe Lys Glu His Trp Ser His Asp Pro Phe Glu Ala Phe 50 5560 Lys Asp Ser Glu Gly Tyr Ile Tyr Ala Arg Gly Ala Gln Asp Met Lys 65 7075 80 Cys Val Ser Ile Gln Tyr Leu Glu Ala Val Arg Arg Leu Lys Val Glu 8590 95 Gly His Arg Phe Pro Arg Thr Ile His Met Thr Phe Val Pro Asp Glu100 105 110 Glu Val Gly Gly His Gln Gly Met Glu Leu Phe Val Gln Arg ProGlu 115 120 125 Phe His Ala Leu Arg Ala Gly Phe Ala Leu Asp Glu Gly IleAla Asn 130 135 140 Pro Thr Asp Ala Phe Thr Val Phe Tyr Ser Glu Arg SerPro Trp Trp 145 150 155 160 Val Arg Val Thr Ser Thr Gly Arg Pro Gly HisAla Ser Arg Phe Met 165 170 175 Glu Asp Thr Ala Ala Glu Lys Leu His LysVal Val Asn Ser Ile Leu 180 185 190 Ala Phe Arg Glu Lys Glu Trp Gln ArgLeu Gln Ser Asn Pro His Leu 195 200 205 Lys Glu Gly Ser Val Thr Ser ValAsn Leu Thr Lys Leu Glu Gly Gly 210 215 220 Val Ala Tyr Asn Val Ile ProAla Thr Met Ser Ala Ser Phe Asp Phe 225 230 235 240 Arg Val Ala Pro AspVal Asp Phe Lys Ala Phe Glu Glu Gln Leu Gln 245 250 255 Ser Trp Cys GlnAla Ala Gly Glu Gly Val Thr Leu Glu Phe Ala Gln 260 265 270 Lys Trp MetHis Pro Gln Val Thr Pro Thr Asp Asp Ser Asn Pro Trp 275 280 285 Trp AlaAla Phe Ser Arg Val Cys Lys Asp Met Asn Leu Thr Leu Glu 290 295 300 ProGlu Ile Met Pro Ala Ala Thr Asp Asn Arg Tyr Ile Arg Ala Val 305 310 315320 Gly Val Pro Ala Leu Gly Phe Ser Pro Met Asn Arg Thr Pro Val Leu 325330 335 Leu His Asp His Asp Glu Arg Leu His Glu Ala Val Phe Leu Arg Gly340 345 350 Val Asp Ile Tyr Thr Arg Leu Leu Pro Ala Leu Ala Ser Val ProAla 355 360 365 Leu Pro Ser Asp Ser 370 3 9704 DNA Homo sapiens 3gctgcatgac cacagggatt ggtgggaaat ccagggtctg gacagccaag ccaaggaagt 60caggaaccta gagggtatgg ggaacgcgat ttaacaatta gccagcattg gccgggcgca 120gtggctcaca cctgtaatcc cagcactttg ggaggccgag gcaggcggat cacgaggtca 180ggagatcgag accatcctga ctaacacggt gaaaacccgt ctctactaaa aatacaaaaa 240attagccgag cgtggtggcg ggtgacttta gtcccagcta ctcagtaggc tgaggcagga 300gaatggtgtg aacccgggag gcggagcttg cagtgagcca agaccgagat cacaccactg 360cactccaccc tgggtgacaa agcgagtgag actccgtctc aaaaaaaaaa aaaaaaaaaa 420aaaaaaaaaa aaaaaaaaaa aaaaaaaaca ttagctgggc actgtggctc acatctgtaa 480tcccagcacc ttgggaggcc aaggcgggtg gatcacctga ggtcaggagt tcaagaccac 540cctggccaac atgacaaacc ctgtctctac taaaaataca aaaattagcc cagcgtggtg 600gcacgcacct gtaatcccag ctactctgga ggctgaggca ggagaatcac ttgaacccag 660gaggcggagt tttcagcgag ccgagaagga gccactgcac tccagcctgg gcagcagagt 720gagactccat ctcaaaaaaa taaatagcta aataattagc cagcattgtt atgagttaaa 780gtctatttgc ccgcatgaat aaataggtaa ataattagcc agcattgttg tgagttaaaa 840tctatttgcc cgcatgacag agtgagactc tgtatcaaaa aaataactaa ataaagaatt 900atccagcatt gttatgagtt aaagtccatt tgcccccatg ttatgtgtga gcagccaaga 960cttaaacctc aggaaaggtg ggacagaacc cttcccacag cgtgcctcct tggcctagag 1020attgaagtct ttgtccctca cccttcccca agctgactca gcccctggag cagagggcag 1080acctggctgg gagtacaaag ggcagctggg gcacaagtgg gcaactgcag ctgtggcctg 1140cagggggcca gtggtacacc tgtgcctgtt tagcctcccc ctctggtggc tgcagaagag 1200ccagtttcct cacactgtcc atccagggtc acaattacat ccattcacag tgacttcatc 1260acacccaccc accatctcac actgtcacat acacaatcat atccactgat agactgcaca 1320cgcagtggca cgcttaaacc gtcacacgtg ctcttgtcca tgcattcatt cccattctag 1380gcactgtccg ggctcggcac ggccccgggc agaaccttgc aggaagtgga gctcacagcc 1440tcctggagtt cagtgtgggc agacagacat tggccaataa cttcagtaca agtggagctg 1500aggcgtcagg gaggacctcc ccgaggggct gaggcctgca gtggggagcc gttggagact 1560tgccgaggag ggcgagggcg caggcccagg gctttgcagc tctgcatctt gagagcctcg 1620gggcggcccc ctttcctccc gccctatccg ggggctgaag gaggaggcgc ccttagggga 1680cgggaccgtc ctgagctccc ggcgcatacc tgggggcagg agtggcaggc gtgtcgtgtg 1740gggcggggcg agcctgtcag agcagggcca gcccggagct cgcaactcgc ggggcggcgc 1800tggccgcggc ggccgctgcc cggggacggg atccggatct aatcctccag taatctcgct 1860gaggcccgaa ccagaggcgg gcggggacat ccgcgccgac gcggccgctg gcgccgggac 1920ggccctcact gacggtcttc ggtctccgcc ccgacatccg gcctcggcca cgtggtgggc 1980ggaccggggc ggtcctgagc ctgcgacctc gcaggcgacc tcgctggacc ctaagtccag 2040gccacagtca gggaagggcg ctgagaggcg agcgtgagcc cagcgacagg agagtgaggt 2100gggggccctg gggagggata gagggactgg ggctccgtgg cttgaaagcc gggcaactgg 2160gaggcgttgg ggtttttctt gtttgttttt tgtttttgtt tttgcctttt ttttttttta 2220ggagggcggg gggagtacaa gtctgggttc aaaccttgct cagctactct atgagctgtc 2280cttgaacctc tctgagcctc tcagctttct cctctgtaaa gtgggcattc tgagcacaaa 2340cttcatgggg ctcttttggg gattaaataa ggaaatgtgc tggaagcaga cagcccagcg 2400cctgaaacag aatgggtgct ccttaatggg ggctccgaaa cacggtatcc tacccctgtg 2460ggaagtccgg gagccgccgt ggggacaggc tgtgtgcagg agctcaccat ttccagggtc 2520ttggaggggt agttagccat tcactttgcc cccagctcac cacgcgcagc gccatgacca 2580gcaagggtcc cgaggaggag cacccatcgg tgacgctctt ccgccagtac ctgcgtatcc 2640gcactgtcca gcccaagcct gactatggtg agaagacggt ggttccagag cctgtgacgg 2700ggcctaaggg acggggactg tgctctaaac cagcctccaa cccctgtcac ccagctgagc 2760cccactctgc tgtcccaaat ggctccccaa cccctccagc cattccccaa gtaaatagac 2820tgaggcagcc cctccaggtt agggaggaac cctttcccca gagactctgc tgctgaccaa 2880ggttactcct ggcagctggt taaagaaaaa cttcacctca ctctccaggg caggagtggt 2940gggggaagcc tgaggcagcc acagggaaag gagaggccct ccagaagccc actggggctg 3000gacaaaggcc acagccctta gggagtcaag cttggtggct agggcctggg aggtggctcc 3060tgcctgttat cccagcactt caggaggttg aggctggcag attgcttggg cccaggagtt 3120caagactagc ttgagcaaca tggcaagact ctgtctctac agaaaaaata caaaaattag 3180tcaggaatgg tggcacacct gtagtcccag ctactccaga ggttgaggtg ggaggatcgc 3240ttgagcctgg gaggttgagg ctgcagtgag ccgagatcgc accacttcac tcctgccttg 3300gtgacagagt gagaccctgt ctcaaaaaaa aaaaaaaaaa aaggaaaaga aaaaaaaaaa 3360acttagtggc tgggaattgt gtacatgggt ccaaattcct cctctgtgat taatcagctg 3420agagatggtg ggtgaatctc ttcatgtctc tgtgccatag tttcccatat ttaaggaaga 3480taacaccttc ctccaaccct gtgtccagac atccccctgg acttccagaa agggtcactg 3540agtagccaaa aatatcttct ttcttgggga tggaaatgca agcatctctg agggatatgg 3600agtgtgtcgg ggaggcagca gcccatttct gggtatgctc cactctccgg gctgcctggg 3660ctggtgggaa gctgtgggta ggcagaagca gccccaagac actctgtgcc tccaggagct 3720gctgtggctt tctttgagga gacagcccgc cagctgggcc tgggctgtca gaaagtagag 3780gtgagcctgg ggccctaagc ggggaaggga ggtgggcctg ggcacttcct caccctgctc 3840agaccaccta ccctcctgac catctccagg tggcacctgg ctatgtggtg accgtgttga 3900cctggccagg caccaaccct acactctcct ccatcttgct caactcccac acggatgtgg 3960tgcctgtctt caaggtgtgt aaggggctgg ggaggtgggc agtgcaggcc ttggggacag 4020acatgatgca gaccccagga ttcaacctca agttgctcat ggtcctggcc ccagtcctga 4080cactaactct caacatcctt atgacattac accactcaag cagccttcat ccagcagcaa 4140gttctgggcc agagtggggt ggggactggg gggtgggaag caggagacag caatggggga 4200tggcaatcag ctgccttctt cagcccccgt ctttcctctc ccaccactcc acctgtcact 4260ccaaccctat ggtgggctcc tagggcaggg ccactgttga ccagagtgga ttaatggcta 4320aatttggggt ttgggcccct cttcccatcc ctgcccccag gaacattgga gtcacgaccc 4380ctttgaggcc ttcaaggatt ctgagggcta catctatgcc aggggtgccc aggacatgaa 4440gtgcgtcagc atccagtgag tgtcctccat tcctactcct ccacaatgtc cccactggtc 4500cagtggattg aagcaggacc tgagggggtg attggagaaa ctcaaggcca aggaacaccg 4560tgacctcttg gacaggaact actgccatga ccattgcatg gatagggaga ttcagaccag 4620agaggggcag ggactttctg gagtccctat cagggtgtgg cagggtaaag tccaggacac 4680aggactccag cctgctggcc ctgcctgtgg ggccagcctg cgcatctggt ggctccccca 4740gcacctggct tatgccccct caggtacctg gaagctgtga ggaggctgaa ggtggagggc 4800caccggttcc ccagaaccat ccacatgacc tttgtgcctg gtaggagtgg ctcagatacc 4860tttgggaaag gggagggtgg ggcggggcag cctcctcatc tcacgtccct gctgctttta 4920cagatgagga ggttgggggt caccaaggca tggagctgtt cgtgcagcgg cctgagttcc 4980acgccctgag ggcaggcttt gccctggatg agggtgagca ggttggcaag ccaatgagca 5040gccaggcagg gagtaggagg ctgctagtgg ggactgagct gctccaccct ctgaaccccc 5100tttccctcct caggcatagc caatcccact gatgccttca ctgtctttta tagtgagcgg 5160agtccctggt gtaagtatga gcttggaggg agggctcact ctacaggcgg gaggctaggc 5220cagaaagggc acggtcctat gcagggttgc acagcaaagt tgaggcctga gaaggccttg 5280aacccagggc ctctacctcc cagctctttc ctatctgagc ttctctgagg gcaagccctg 5340aatgggcaga aaccagctgt atgctacggg ccctgagtgg ggacaggacc ctgccagagg 5400agcctggaat gagggggaga cctgggccca ccccaggctg attgtgtctc cagcccctca 5460ggctgaagac actgccttcc ccctacacct ccccaggggt gcgggttacc agcactggga 5520ggccaggcca tgcctcacgc ttcatggagg acacagcagc agagaagctg gtacgtggca 5580ccccaggagg gagtctggga gttcaggagg ctctatcctg aggccactgt cccatttaac 5640ctcatattct catagcacaa ggttgtaaac tccatcctgg cattccggga gaaggaatgg 5700cagaggtgag gcagcctggg aggcagtggg gtggctctgg gaggcggtac cacagaggat 5760agagtctgag ccacctcttt tatctgttgc tgccgctacc ctgcccccac accacaggct 5820gcagtcaaac ccccacctga aagaggggtc cgtgacctcc gtgaacctga ctaagctaga 5880gggtggcgtg gcctataacg tgatacctgc caccatgagc gccagctttg acttccgtgt 5940ggcaccggat gtggacttca aggtgccacc tccacctggg tttggaggag ggatcctggg 6000tcctcagtct tgtcctagag gcctctggaa agcctgaagg atcagctcgt ctcccttctc 6060ttaggctttt gaggagcagc tgcagagctg gtgccaggca gctggcgagg gggtcaccct 6120agagtttgct caggtatgga cttgggacat gtgatgggag agtgtgggag ccgggggaga 6180cccaagtgtg caacagtgga gtgtgtgctt ggtgtgtctg catatgtctg ggcatttctt 6240tatctgtgac agacacattt tattccaaca agcattcatt gtagaggcca ctgtgggtgc 6300tggggaatgc tgtggggagt aaaattaggc acagttcatg cccttgtatg gtgaaacggg 6360gagatataaa tcaaacattt atgtgatatt acttttttct gagagaatct cactccgtca 6420cccaggctgc agtgcagtgg cacaatctcg gctcacctcc gcctcccggg ttcaagcaat 6480tcttgtgcct cagcctccag agtagttggg attacaggca cctgccacca cgcccagcta 6540atttttgcat ttttagtaga gacagtgttt caccatgttg gccaggcttg tctcgaactc 6600ctggcctcaa gtgatccacc caccttggcc tcgcaaaatg ctgggattac aggcatgagc 6660cactgcgccc agccgtactt tcatataacc catgtggtac aggaaagggt ggccccttgc 6720actctgaaaa cctgtaactg gagtatccaa ctagtctgag aggtctgggg gagccatctt 6780gaggaagggg cacttgggct aggatctgaa ggatggacag gaggtaagta gacggagggt 6840gggaaggtcc cagacctagg acatttgagg ggctgaaaga ggacctgtgg ctggactggc 6900tacccagatg tctgggtagg tgaaggagtg ggggtgggga ggtgttatgt actaggcaca 6960gcccactcta tgggaaatag ggcaagatgc ccaggcccat gtcctgatcc tgccattctt 7020cctgtccctc agaagtggat gcacccccaa gtgacaccta ctgatgactc aaacccttgg 7080tgggcagctt ttagccgggt ctgcaaggat atgtgagcac gctggccagc tctcctcaca 7140gcccagcccc ctactcctct ccttcctgct gccccctccc ttctccctcc ttctcccacc 7200tctttcccac cttcctttgc cccttcaatt cttcgctttc tccctcccca ttcatcaggc 7260tctttctcct acaggaacct cactctggag cctgagatca tgcctgctgc cactgacaac 7320cgctatatcc gcgcggtgag ccacttgcat atagtgcctg ggcagtggac tgggcctgag 7380tgctggcttt tccctaacgg ctcttcctca cccctgcagg tgggggtccc agctctaggc 7440ttctcaccca tgaaccgcac acctgtgctg ctgcacgacc acgatgaacg gctgcatgag 7500gctgtgttcc tccgtggggt ggacatatat acacgcctgc tgcctgccct tgccagtgtg 7560cctgccctgc ccagtgacag ctgagccctg gaactcctaa acctttgccc ctggggcttc 7620catcccaacc agtgccaagg acctcctctt cccccttcca aataataaag tctatggaca 7680gggctgtctc tgaagtacta acacaaggac actcgtggag caagaatttt ccttttcctg 7740gggacatgtt accatctcca tttcacagat gaggaaactg agcctggctg ttagcacttc 7800cccactaccc cacactgctc tgtgcccctt gacacagcac acccattcag taccatccag 7860ccatgtctgt gcctagcaag aaagggccac agttcctatt tgagtggcca ccatacttag 7920ttctgaccta tcagggattc cattcccatt aaagagggat actaaggacc tcaggaacca 7980ctcccatctt cctgggtgta catctgggat cctgagacag taccagaata gcaccagctg 8040ggcccctgct agatgagggg caggcagagg gccaacggtg actgctggct cctgtcaaaa 8100cctgtacacc cttgtgttgg cagcaggggc cacagagggg cagggtccct ggtagactag 8160gtcagttcat cttagaggcc tcagcaccct ggatctgtgt gtgcagaggc ccaggaactg 8220ggctttcatc tcagccttgc taggaccccc aggtagtacc aagagtaaac tatggcccca 8280gtagcagagc ctgatctagc cagatctgct ctatcctgtt ctgacttccc tgagcatggg 8340gcaggagaga cagggctggg gtgggatagt tggatttttt aagtttctag ttgtagccag 8400aagtccagag cctggctctg ggctgcaggc ttagtactaa tagaaataac aatcactcct 8460gctcacagtt gacaaggagc caggacttga ctggcttttt tttttttttt ttttttttga 8520gatggagtct ttctctgtcg cccaggctgg cgtgcagtgg cgcgatctcg gctcactgca 8580aactccgctt cctgggttca cgccattctc ctgcctcagt ttcctgagta gttgggacta 8640caggcccccg ccaccacgcc cagctttttg tatttttagt agagacgggg tttcacctcc 8700gcctcccagg ttcaagggat tctcctgcct cagcctccca agtagctggg actacatgcg 8760cgtgccacca cggccggcta atttttgtat ttttagtaga gacggtttca ccacgttgaa 8820caggatgatt tcgatctctt gacctcaggg gatccgcctg cctcggcctc ccaaagtgct 8880ggtgagaggt gacagcgtgc tggcagtcct cacagccctc gctcgctctc cccgcctcct 8940ctgcctcggc tcccactttg gtggcacttg aggagccctt cagcccaccg ctgcactgta 9000ggaacccctt tctgggctgg ccaaggccag agccggctcc ctcagttcgc agggaggtgt 9060ggagggagag gcgcgagcgg gaaccggggc tgcccgccgc gcttgcgggc cagctggagt 9120tccgggtggg cgtgggtttg gcgggccccg cactcgcact cggagcagcc ggccggccct 9180gccgtccccg ccgtccccgg gcaatgaggg gcttagcacc cgggccagtg gctgcggagg 9240gtgtactggg tcccccagca gtgccaggcc accggcgctg ctctcgattt ctcaccgggt 9300cttagctgcc ttcccgcggg tcagggtttg ggacctgcag cccaccatgc cttgagccct 9360cccaccccct ccactggctc ccgtgcggcc ccagcctccc ccatgagcgc cgccccccgc 9420tccacggcac ccagtcccat ccaccaccca agggctgagg agtgcgggct cacggagcag 9480gactggcagg cagctccacc tgcagccccg gtgcgggatc cactgggtga agccagctgg 9540gctcctgagt ctggtgggga cgtggagaac ctttatgtct agctcaggga ttgtaaatac 9600accaatcggc attctgtatc tagctcaagg tttgtaaaca caccaatcag caccctgtgt 9660ctagctcagg gtttgtgaat acaccaatgg acactctgta tcta 9704 4 408 PRT Homosapiens 4 Met Thr Ser Lys Gly Pro Glu Glu Glu His Pro Ser Val Thr LeuPhe 1 5 10 15 Arg Gln Tyr Leu Arg Ile Arg Thr Val Gln Pro Lys Pro AspTyr Gly 20 25 30 Ala Ala Val Ala Phe Phe Glu Glu Thr Ala Arg Gln Leu GlyLeu Gly 35 40 45 Cys Gln Lys Val Glu Val Ala Pro Gly Tyr Val Val Thr ValLeu Thr 50 55 60 Trp Pro Gly Thr Asn Pro Thr Leu Ser Ser Ile Leu Leu AsnSer His 65 70 75 80 Thr Asp Val Val Pro Val Phe Lys Glu His Trp Ser HisAsp Pro Phe 85 90 95 Glu Ala Phe Lys Asp Ser Glu Gly Tyr Ile Tyr Ala ArgGly Ala Gln 100 105 110 Asp Met Lys Cys Val Ser Ile Gln Tyr Leu Glu AlaVal Arg Arg Leu 115 120 125 Lys Val Glu Gly His Arg Phe Pro Arg Thr IleHis Met Thr Phe Val 130 135 140 Pro Asp Glu Glu Val Gly Gly His Gln GlyMet Glu Leu Phe Val Gln 145 150 155 160 Arg Pro Glu Phe His Ala Leu ArgAla Gly Phe Ala Leu Asp Glu Gly 165 170 175 Ile Ala Asn Pro Thr Asp AlaPhe Thr Val Phe Tyr Ser Glu Arg Ser 180 185 190 Pro Trp Trp Val Arg ValThr Ser Thr Gly Arg Pro Gly His Ala Ser 195 200 205 Arg Phe Met Glu AspThr Ala Ala Glu Lys Leu His Lys Val Val Asn 210 215 220 Ser Ile Leu AlaPhe Arg Glu Lys Glu Trp Gln Arg Leu Gln Ser Asn 225 230 235 240 Pro HisLeu Lys Glu Gly Ser Val Thr Ser Val Asn Leu Thr Lys Leu 245 250 255 GluGly Gly Val Ala Tyr Asn Val Ile Pro Ala Thr Met Ser Ala Ser 260 265 270Phe Asp Phe Arg Val Ala Pro Asp Val Asp Phe Lys Ala Phe Glu Glu 275 280285 Gln Leu Gln Ser Trp Cys Gln Ala Ala Gly Glu Gly Val Thr Leu Glu 290295 300 Phe Ala Gln Lys Trp Met His Pro Gln Val Thr Pro Thr Asp Asp Ser305 310 315 320 Asn Pro Trp Trp Ala Ala Phe Ser Arg Val Cys Lys Asp MetAsn Leu 325 330 335 Thr Leu Glu Pro Glu Ile Met Pro Ala Ala Thr Asp AsnArg Tyr Ile 340 345 350 Arg Ala Val Gly Val Pro Ala Leu Gly Phe Ser ProMet Asn Arg Thr 355 360 365 Pro Val Leu Leu His Asp His Asp Glu Arg LeuHis Glu Ala Val Phe 370 375 380 Leu Arg Gly Val Asp Ile Tyr Thr Arg LeuLeu Pro Ala Leu Ala Ser 385 390 395 400 Val Pro Ala Leu Pro Ser Asp Ser405 5 601 DNA Homo sapiens 5 ttcaagacca ccctggccaa catgacaaac cctgtctctactaaaaatac aaaaattagc 60 ccagcgtggt ggcacgcacc tgtaatccca gctactctggaggctgaggc aggagaatca 120 cttgaaccca ggaggcggag ttttcagcga gccgagaaggagccactgca ctccagcctg 180 ggcagcagag tgagactcca tctcaaaaaa ataaatagctaaataattag ccagcattgt 240 tatgagttaa agtctatttg cccgcatgaa taaataggtaaataattagc cagcattgtt 300 rtgagttaaa atctatttgc ccgcatgaca gagtgagactctgtatcaaa aaaataacta 360 aataaagaat tatccagcat tgttatgagt taaagtccatttgcccccat gttatgtgtg 420 agcagccaag acttaaacct caggaaaggt gggacagaacccttcccaca gcgtgcctcc 480 ttggcctaga gattgaagtc tttgtccctc acccttccccaagctgactc agcccctgga 540 gcagagggca gacctggctg ggagtacaaa gggcagctggggcacaagtg ggcaactgca 600 g 601 6 601 DNA Homo sapiens 6 gtagttagccattcactttg cccccagctc accacgcgca gcgccatgac cagcaagggt 60 cccgaggaggagcacccatc ggtgacgctc ttccgccagt acctgcgtat ccgcactgtc 120 cagcccaagcctgactatgg tgagaagacg gtggttccag agcctgtgac ggggcctaag 180 ggacggggactgtgctctaa accagcctcc aacccctgtc acccagctga gccccactct 240 gctgtcccaaatggctcccc aacccctcca gccattcccc aagtaaatag actgaggcag 300 yccctccaggttagggagga accctttccc cagagactct gctgctgacc aaggttactc 360 ctggcagctggttaaagaaa aacttcacct cactctccag ggcaggagtg gtgggggaag 420 cctgaggcagccacagggaa aggagaggcc ctccagaagc ccactggggc tggacaaagg 480 ccacagcccttagggagtca agcttggtgg ctagggcctg ggaggtggct cctgcctgtt 540 atcccagcacttcaggaggt tgaggctggc agattgcttg ggcccaggag ttcaagacta 600 g 601 7 601DNA Homo sapiens 7 tgccttcttc agcccccgtc tttcctctcc caccactccacctgtcactc caaccctatg 60 gtgggctcct agggcagggc cactgttgac cagagtggattaatggctaa atttggggtt 120 tgggcccctc ttcccatccc tgcccccagg aacattggagtcacgacccc tttgaggcct 180 tcaaggattc tgagggctac atctatgcca ggggtgcccaggacatgaag tgcgtcagca 240 tccagtgagt gtcctccatt cctactcctc cacaatgtccccactggtcc agtggattga 300 mgcaggacct gagggggtga ttggagaaac tcaaggccaaggaacaccgt gacctcttgg 360 acaggaacta ctgccatgac cattgcatgg atagggagattcagaccaga gaggggcagg 420 gactttctgg agtccctatc agggtgtggc agggtaaagtccaggacaca ggactccagc 480 ctgctggccc tgcctgtggg gccagcctgc gcatctggtggctcccccag cacctggctt 540 atgccccctc aggtacctgg aagctgtgag gaggctgaaggtggagggcc accggttccc 600 c 601 8 601 DNA Homo sapiens 8 tcactccaaccctatggtgg gctcctaggg cagggccact gttgaccaga gtggattaat 60 ggctaaatttggggtttggg cccctcttcc catccctgcc cccaggaaca ttggagtcac 120 gacccctttgaggccttcaa ggattctgag ggctacatct atgccagggg tgcccaggac 180 atgaagtgcgtcagcatcca gtgagtgtcc tccattccta ctcctccaca atgtccccac 240 tggtccagtggattgaagca ggacctgagg gggtgattgg agaaactcaa ggccaaggaa 300 yaccgtgacctcttggacag gaactactgc catgaccatt gcatggatag ggagattcag 360 accagagaggggcagggact ttctggagtc cctatcaggg tgtggcaggg taaagtccag 420 gacacaggactccagcctgc tggccctgcc tgtggggcca gcctgcgcat ctggtggctc 480 ccccagcacctggcttatgc cccctcaggt acctggaagc tgtgaggagg ctgaaggtgg 540 agggccaccggttccccaga accatccaca tgacctttgt gcctggtagg agtggctcag 600 a 601 9 601DNA Homo sapiens 9 agggagattc agaccagaga ggggcaggga ctttctggagtccctatcag ggtgtggcag 60 ggtaaagtcc aggacacagg actccagcct gctggccctgcctgtggggc cagcctgcgc 120 atctggtggc tcccccagca cctggcttat gccccctcaggtacctggaa gctgtgagga 180 ggctgaaggt ggagggccac cggttcccca gaaccatccacatgaccttt gtgcctggta 240 ggagtggctc agataccttt gggaaagggg agggtggggcggggcagcct cctcatctca 300 ygtccctgct gcttttacag atgaggaggt tgggggtcaccaaggcatgg agctgttcgt 360 gcagcggcct gagttccacg ccctgagggc aggctttgccctggatgagg gtgagcaggt 420 tggcaagcca atgagcagcc aggcagggag taggaggctgctagtgggga ctgagctgct 480 ccaccctctg aacccccttt ccctcctcag gcatagccaatcccactgat gccttcactg 540 tcttttatag tgagcggagt ccctggtgta agtatgagcttggagggagg gctcactcta 600 c 601 10 601 DNA Homo sapiens 10 ccctgagggcaggctttgcc ctggatgagg gtgagcaggt tggcaagcca atgagcagcc 60 aggcagggagtaggaggctg ctagtgggga ctgagctgct ccaccctctg aacccccttt 120 ccctcctcaggcatagccaa tcccactgat gccttcactg tcttttatag tgagcggagt 180 ccctggtgtaagtatgagct tggagggagg gctcactcta caggcgggag gctaggccag 240 aaagggcacggtcctatgca gggttgcaca gcaaagttga ggcctgagaa ggccttgaac 300 ycagggcctctacctcccag ctctttccta tctgagcttc tctgagggca agccctgaat 360 gggcagaaaccagctgtatg ctacgggccc tgagtgggga caggaccctg ccagaggagc 420 ctggaatgagggggagacct gggcccaccc caggctgatt gtgtctccag cccctcaggc 480 tgaagacactgccttccccc tacacctccc caggggtgcg ggttaccagc actgggaggc 540 caggccatgcctcacgcttc atggaggaca cagcagcaga gaagctggta cgtggcaccc 600 c 601 11 601DNA Homo sapiens 11 cagggcctct acctcccagc tctttcctat ctgagcttctctgagggcaa gccctgaatg 60 ggcagaaacc agctgtatgc tacgggccct gagtggggacaggaccctgc cagaggagcc 120 tggaatgagg gggagacctg ggcccacccc aggctgattgtgtctccagc ccctcaggct 180 gaagacactg ccttccccct acacctcccc aggggtgcgggttaccagca ctgggaggcc 240 aggccatgcc tcacgcttca tggaggacac agcagcagagaagctggtac gtggcacccc 300 rggagggagt ctgggagttc aggaggctct atcctgaggccactgtccca tttaacctca 360 tattctcata gcacaaggtt gtaaactcca tcctggcattccgggagaag gaatggcaga 420 ggtgaggcag cctgggaggc agtggggtgg ctctgggaggcggtaccaca gaggatagag 480 tctgagccac ctcttttatc tgttgctgcc gctaccctgcccccacacca caggctgcag 540 tcaaaccccc acctgaaaga ggggtccgtg acctccgtgaacctgactaa gctagagggt 600 g 601 12 601 DNA Homo sapiens 12 ccagcactgggaggccaggc catgcctcac gcttcatgga ggacacagca gcagagaagc 60 tggtacgtggcaccccagga gggagtctgg gagttcagga ggctctatcc tgaggccact 120 gtcccatttaacctcatatt ctcatagcac aaggttgtaa actccatcct ggcattccgg 180 gagaaggaatggcagaggtg aggcagcctg ggaggcagtg gggtggctct gggaggcggt 240 accacagaggatagagtctg agccacctct tttatctgtt gctgccgcta ccctgccccc 300 rcaccacaggctgcagtcaa acccccacct gaaagagggg tccgtgacct ccgtgaacct 360 gactaagctagagggtggcg tggcctataa cgtgatacct gccaccatga gcgccagctt 420 tgacttccgtgtggcaccgg atgtggactt caaggtgcca cctccacctg ggtttggagg 480 agggatcctgggtcctcagt cttgtcctag aggcctctgg aaagcctgaa ggatcagctc 540 gtctcccttctcttaggctt ttgaggagca gctgcagagc tggtgccagg cagctggcga 600 g 601 13 601DNA Homo sapiens 13 ctggagcctg agatcatgcc tgctgccact gacaaccgctatatccgcgc ggtgagccac 60 ttgcatatag tgcctgggca gtggactggg cctgagtgctggcttttccc taacggctct 120 tcctcacccc tgcaggtggg ggtcccagct ctaggcttctcacccatgaa ccgcacacct 180 gtgctgctgc acgaccacga tgaacggctg catgaggctgtgttcctccg tggggtggac 240 atatatacac gcctgctgcc tgcccttgcc agtgtgcctgccctgcccag tgacagctga 300 sccctggaac tcctaaacct ttgcccctgg ggcttccatcccaaccagtg ccaaggacct 360 cctcttcccc cttccaaata ataaagtcta tggacagggctgtctctgaa gtactaacac 420 aaggacactc gtggagcaag aattttcctt ttcctggggacatgttacca tctccatttc 480 acagatgagg aaactgagcc tggctgttag cacttccccactaccccaca ctgctctgtg 540 ccccttgaca cagcacaccc attcagtacc atccagccatgtctgtgcct agcaagaaag 600 g 601 14 601 DNA Human 14 agagggccaacggtgactgc tggctcctgt caaaacctgt acacccttgt gttggcagca 60 ggggccacagaggggcaggg tccctggtag actaggtcag ttcatcttag aggcctcagc 120 accctggatctgtgtgtgca gaggcccagg aactgggctt tcatctcagc cttgctagga 180 cccccaggtagtaccaagag taaactatgg ccccagtagc agagcctgat ctagccagat 240 ctgctctatcctgttctgac ttccctgagc atggggcagg agagacaggg ctggggtggg 300 rtagttggattttttaagtt tctagttgta gccagaagtc cagagcctgg ctctgggctg 360 caggcttagtactaatagaa ataacaatca ctcctgctca cagttgacaa ggagccagga 420 cttgactggctttttttttt tttttttttt tttgagatgg agtctttctc tgtcgcccag 480 gctggcgtgcagtggcgcga tctcggctca ctgcaaactc cgcttcctgg gttcacgcca 540 ttctcctgcctcagtttcct gagtagttgg gactacaggc ccccgccacc acgcccagct 600 t 601

That which is claimed is:
 1. An isolated peptide consisting of an amino acid sequence selected from the group consisting of: (a) an amino acid sequence shown in SEQ ID NO: 2; (b) an amino acid sequence of an allelic variant of an amino acid sequence shown in SEQ ID NO: 2, wherein said allelic variant is encoded by a nucleic acid molecule that hybridizes under stringent conditions to the opposite strand of a nucleic acid molecule shown in SEQ ID NOS: 1 or 3; (c) an amino acid sequence of an ortholog of an amino acid sequence shown in SEQ ID NO: 2, wherein said ortholog is encoded by a nucleic acid molecule that hybridizes under stringent conditions to the opposite strand of a nucleic acid molecule shown in SEQ ID NOS: 1 or 3; and (d) a fragment of an amino acid sequence shown in SEQ ID NO: 2, wherein said fragment comprises at least 10 contiguous amino acids.
 2. An isolated peptide comprising an amino acid sequence selected from the group consisting of: (a) an amino acid sequence shown in SEQ ID NO: 2; (b) an amino acid sequence of an allelic variant of an amino acid sequence shown in SEQ ID NO: 2, wherein said allelic variant is encoded by a nucleic acid molecule that hybridizes under stringent conditions to the opposite strand of a nucleic acid molecule shown in SEQ ID NOS: 1 or 3; (c) an amino acid sequence of an ortholog of an amino acid sequence shown in SEQ ID NO: 2, wherein said ortholog is encoded by a nucleic acid molecule that hybridizes under stringent conditions to the opposite strand of a nucleic acid molecule shown in SEQ ID NOS: 1 or 3; and (d) a fragment of an amino acid sequence shown in SEQ ID NO: 2, wherein said fragment comprises at least 10 contiguous amino acids.
 3. An isolated antibody that selectively binds to a peptide of claim
 2. 4. An isolated nucleic acid molecule consisting of a nucleotide sequence selected from the group consisting of: (a) a nucleotide sequence that encodes an amino acid sequence shown in SEQ ID NO: 2; (b) a nucleotide sequence that encodes of an allelic variant of an amino acid sequence shown in SEQ ID NO: 2, wherein said nucleotide sequence hybridizes under stringent conditions to the opposite strand of a nucleic acid molecule shown in SEQ ID NOS: 1 or 3; (c) a nucleotide sequence that encodes an ortholog of an amino acid sequence shown in SEQ ID NO: 2, wherein said nucleotide sequence hybridizes under stringent conditions to the opposite strand of a nucleic acid molecule shown in SEQ ID NOS: 1 or 3; (d) a nucleotide sequence that encodes a fragment of an amino acid sequence shown in SEQ ID NO: 2, wherein said fragment comprises at least 10 contiguous amino acids; and (e) a nucleotide sequence that is the complement of a nucleotide sequence of (a)-(d).
 5. An isolated nucleic acid molecule comprising a nucleotide sequence selected from the group consisting of: (a) a nucleotide sequence that encodes an amino acid sequence shown in SEQ ID NO: 2; (b) a nucleotide sequence that encodes of an allelic variant of an amino acid sequence shown in SEQ ID NO: 2, wherein said nucleotide sequence hybridizes under stringent conditions to the opposite strand of a nucleic acid molecule shown in SEQ ID NOS: 1 or 3; (c) a nucleotide sequence that encodes an ortholog of an amino acid sequence shown in SEQ ID NO: 2, wherein said nucleotide sequence hybridizes under stringent conditions to the opposite strand of a nucleic acid molecule shown in SEQ ID NOS: 1 or 3; (d) a nucleotide sequence that encodes a fragment of an amino acid sequence shown in SEQ ID NO: 2, wherein said fragment comprises at least 10 contiguous amino acids; and (e) a nucleotide sequence that is the complement of a nucleotide sequence of (a)-(d).
 6. A gene chip comprising a nucleic acid molecule of claim
 5. 7. A transgenic non-human animal comprising a nucleic acid molecule of claim
 5. 8. A nucleic acid vector comprising a nucleic acid molecule of claim
 5. 9. A host cell containing the vector of claim
 8. 10. A method for producing any of the peptides of claim 1 comprising introducing a nucleotide sequence encoding any of the amino acid sequences in (a)-(d) into a host cell, and culturing the host cell under conditions in which the peptides are expressed from the nucleotide sequence.
 11. A method for producing any of the peptides of claim 2 comprising introducing a nucleotide sequence encoding any of the amino acid sequences in (a)-(d) into a host cell, and culturing the host cell under conditions in which the peptides are expressed from the nucleotide sequence.
 12. A method for detecting the presence of any of the peptides of claim 2 in a sample, said method comprising contacting said sample with a detection agent that specifically allows detection of the presence of the peptide in the sample and then detecting the presence of the peptide.
 13. A method for detecting the presence of a nucleic acid molecule of claim 5 in a sample, said method comprising contacting the sample with an oligonucleotide that hybridizes to said nucleic acid molecule under stringent conditions and determining whether the oligonucleotide binds to said nucleic acid molecule in the sample.
 14. A method for identifying a modulator of a peptide of claim 2, said method comprising contacting said peptide with an agent and determining if said agent has modulated the function or activity of said peptide.
 15. The method of claim 14, wherein said agent is administered to a host cell comprising an expression vector that expresses said peptide.
 16. A method for identifying an agent that binds to any of the peptides of claim 2, said method comprising contacting the peptide with an agent and assaying the contacted mixture to determine whether a complex is formed with the agent bound to the peptide.
 17. A pharmaceutical composition comprising an agent identified by the method of claim 16 and a pharmaceutically acceptable carrier therefor.
 18. A method for treating a disease or condition mediated by a human enzyme protein, said method comprising administering to a patient a pharmaceutically effective amount of an agent identified by the method of claim
 16. 19. A method for identifying a modulator of the expression of a peptide of claim 2, said method comprising contacting a cell expressing said peptide with an agent, and determining if said agent has modulated the expression of said peptide.
 20. An isolated human enzyme peptide having an amino acid sequence that shares at least 70% homology with an amino acid sequence shown in SEQ ID NO:
 2. 21. A peptide according to claim 20 that shares at least 90 percent homology with an amino acid sequence shown in SEQ ID NO:
 2. 22. An isolated nucleic acid molecule encoding a human enzyme peptide, said nucleic acid molecule sharing at least 80 percent homology with a nucleic acid molecule shown in SEQ ID NOS: 1 or
 3. 23. A nucleic acid molecule according to claim 22 that shares at least 90 percent homology with a nucleic acid molecule shown in SEQ ID NOS: 1 or
 3. 